bluuebunny/crossref_metadata_2025 huggingface dataset:
Ex vivo real‐time MMP activation in kidney in hyperhomocysteinemia
Authors: Sen Utpal , Metreveli Naira , Vacek Thomas , Hughes William M , Tyagi Suresh C
Published: June 2021

Polycystic kidney disease is the primary cause of renal pathology. Very recent studies have demonstrated a positive correlation between plasma homocysteine (Hcy) levels and development of chronic kidney disease; however, the mechanism of glomerular matrix remodeling in Hcy‐induced renal failure is unclear. Here, we hypothesize that hyperhomocysteinemia (HHcy) induces the activation of matrix metalloproteinases (MMPs) in the kidney, which causes acute renal failure. Therefore, we sought to determine the effect of HHcy on MMP activation in isolated perfused kidney. The kidneys from wild type (C57BL/6J) mice were isolated and perfused with physiological salt solution (PSS) for various time frames containing fluorogenic MMP‐2 and ‐9 substrates in presence or absence of Hcy (200 μM). PSS perfused kidney served as control. After perfusion, fluorescence was measured by KODAK 4000 MM imaging station. The kidney perfused with MMP‐2 and ‐9 substrates for 3 hr, in conjunction with Hcy, showed robust increases in MMP activation. This was measured by increases in fluorescence compared to either substrate or PSS alone. The results from acute and real‐time imaging show that HHcy causes MMP‐2 and ‐9 activation in the kidney, and this activation is related to renovascular remodeling during kidney failure.
Expression of cytochrome C, mitochondrium Ca2+, gonadal hormone, TNF, TGF‐β and Smad in ovaries of diabetes rats
Authors: Wang Shu‐Qiu , Kang Yu‐Ming* , Zhang Dong‐Dong , Qin Wen‐Bo , Liang Yan‐Feng
Published: June 2021

The present study was undertaken to observe effects of Ganleroma lucidum spore on cytochrome C, mitochondrium Ca2+, gonadal hormone, TNF, TGF‐β, Smad and apoptotic in ovaries of diabetes mellitus (NIDDM) rat. 30 female rats were randomly divided control group, model group plus Ganleroma lucidum spore. The results showed that T (5.02±1.68 and 4.58±2.45 vs 10.32±3.73 nmol/L), plasma cytochrome C (1.493±0.485 and 1.398±0.294 vs 2.408±0.392 mg/ml), mitochondrium Ca2+ (2.803±0.188 and 2.389±0.366 vs 3.033±0.137μg/L), TNF (18.028±1.113 and 15.205±1.679 vs 40.084±3.460% ), TGF‐&{beta](16.753±1.327 and13.369±2.258 vs 37.768±3.949%) and Smad (15.287±3.929 and 13.287±3.843 vs 36.250±4.164%) were lower (Ganleroma lucidum spore rats and control group rats vs NIDDM rats, P<0.05) than ovaries tissue from Ganleroma lucidum spore rats and control group rats compared with NIDDM rats. The TNF (18.028±1.113 and 15.205±1.679 vs 40.084±3.460%)TGF‐β(16.753±1.327 and13.369±2.258 vs 37.768±3.949%)Smad (15.287±3.929 and 13.287±3.843 vs 36.250±4.164%) .the estrogen(3.09±2.04 and 4.04±2.27 vs 1.68±1.16 nmmo1/L) were higher in that. Conclusions: The decrease in E2 and increase in T, Cytochrome C and mitochondrium Ca2+, TNF, TGF‐βand Smad may adverse factors to ovaries of NIDDM rats Ganoderma lucidum spore can protect the ovaries of NIDDM rats rats.
PPARα Activation with Fenofibrate Attenuates Diabetic Nephropathy in Zucker Diabetic Fatty Rats
Authors: Li Lingyun , Zhao Xueying
Published: June 2021

Peroxisome proliferator–activated receptors (PPARs) are nuclear transcription factors and play an important role in lipid metabolism, glucose homeostasis and inflammation. PPARα is expressed in the kidney, and its agonists exhibit protective effects in type 2 diabetes. In the present study, we investigated the effect of the PPARα agonist fenofibrate on diabetic nephropathy in Zucker diabetic fatty (ZDF) rats. 12‐week‐old male Zucker lean and Zucker diabetic fatty rats were orally administered with fenofibrate (150mg/kg/day) or vehicle for 10 weeks. Treatment with fenofibrate had little effect on body weight gain and blood glucose in Zucker rats. Histological analysis revealed increased glomerular size and collagen deposition in the kidney of ZDF rats. Fenofibrate markedly reduced diabetic glomerular hypertrophy by 17.1%, attenuated collagen expansion by 48.2%, and improved glomerulosclerosis by 69.1%. Gelatin zymography showed MMP‐9 activity was increased in the plasma of ZDF rats, which was further induced by fenofibrate treatment. Furthermore, immunostaining indicated that the over‐expression of α‐smooth muscle actin in diabetic glomeruli and interstitials was significantly decreased by fenofibrate. In conclusion, activation of PPARα with fenofibrate may protect against diabetic nephropathy by attenuating glomerular hypertrophy, renal fibrosis and glomerulosclerosis in ZDF rats.
Regulation of the eIF2/eIF2B system during alcohol‐induced inhibition of hepatic protein synthesis in rats
Authors: Karinch Anne M , Vary Thomas C
Published: June 2021

Alcohol abuse damages hepatic structure and function. The effect of alcohol ingestion on protein synthesis and initiation factors eIF2 and eIF2B in rat liver was studied 1) after acute and chronic ethanol, 2) in males and females after chronic alcohol intake, and 3) after acute alcohol withdrawal. Protein synthesis was measured by incorporation of [3H]‐phenylalanine into liver protein and eIF2/eIF2B content and phosphorylation were determined by immunoblot. Protein synthesis was inhibited 37% after acute and 30% after chronic (>8 wk) ethanol and was associated with 65% increase in eIF2α phosphorylation in either case. eIF2 content decreased in rats fed alcohol for >12 weeks. Basal protein synthesis was 34% higher in females than in males and alcohol‐induced inhibition was greater in females (34%) than in males (19%). Withdrawal of alcohol reversed eIF2α phosphorylation but not the eIF2 deficit so hepatic protein synthesis was partially restored. Acute alcohol administration did not affect eIF2B content or eIF2Bε phosphorylation in male rats whereas chronic alcohol feeding revealed sex‐differential changes in both. eIF2B content in livers from males increased in the alcohol group but decreased in females; eIF2Bε phosphorylation decreased in males and increased in females. Changes in hepatic protein synthesis induced by alcohol consumption reflect underlying alterations in eIF2/eIF2B. NIH R21AA015349
Oxytocin reduces pressor and tachycardic response to the alarming stress in the infarcted rats
Authors: Szczepanska‐Sadowska Ewa Krystyna , Wsol Agnieszka , Kowalewski Stanislaw , Cudnoch‐Jedrzejewska Agnieszka , Dobruch Jakub
Published: June 2021

It is well known that oxytocin (OT) is one of the neuropeptides involved in regulation of behavioral responses to stress. OT may also play a role in central regulation of blood pressure. The purpose of the study was to investigate the role of OT in central cardiovascular control in the infarcted rats. The experiments were performed on conscious male SD rats subjected either to the myocardial infarction or sham surgery. Both groups were instrumented with the left cerebral ventricle (LCV) for central infusions and with the femoral artery catheter for mean arterial blood pressure (MABP) and heart rate (HR) recording. Four weeks after the cardiac surgery the sham and infarcted rats were divided into two groups receiving either LCV infusions of 0.9% NaCl or OT. MABP and HR were recorded during 40 min of rest and during and after the air jet stress. Resting MABP and HR were not affected by OT or 0.9% NaCl infusions. During saline infusion MABP and HR responses to air jet were significantly greater in the infarcted than in the sham rats. Infusion of OT significantly reduced both responses in the infarcted but not in the sham rats. The results provide evidence that oxytocin is able to normalize exaggerated cardiovascular responses to stress during the post‐infarct state. This study was supported by the Medical University of Warsaw – grants 1MA/NM1/06, 1MA/N/07.
Mapping of spinal cord and brainstem inputs to phrenic motoneurons using transneuronal transport of rabies virus N2C in the cat
Authors: Rice Cory D , Lois James H , Mehta Ajeet S , Mehta Amar S , Kalash Ronny , Yates Bill J
Published: June 2021

Rabies virus N2C can be employed to transneuronally trace the neural pathways regulating activity of a particular muscle. Unlike many transneuronal tracers, rabies virus is not avidly transported by autonomic pathways, and results in little lysis of infected neurons. In the present study, rabies virus N2C was injected into the diaphragm of adult cats, which were euthanized following survival periods of 2.5–6 days. Animals were classified by the extent of infection observed in the CNS. In animals with minimal infection, the labeled spinal neurons were comprised of large cells in the C5–C6 ventral horn (presumed diaphragm motoneurons) as well as smaller presumed interneurons confined mainly to the ventral horn and the region near the central canal. Considerably more infected spinal interneurons were present in most cats, which were distributed throughout the dorsal and ventral horns in all cervical segments as well as the thoracic spinal cord. Brainstem labeling in animals with the least infection was confined to the regions known to contain the medullary and pontine respiratory groups, the retrotrapezoid and raphe nuclei, the medial medullary and pontine reticular formation, and the region ventral to the spinal trigeminal nucleus. In animals with intermediate levels of infection, the number of labeled neurons in the respiratory groups and medial reticular formation was considerably larger, and infected cells were also evident in the lateral vestibular nucleus, locus coeruleus, and the cerebellar fastigial nucleus. Infected cells in the medial reticular formation and vestibular and fastigial nuclei likely have roles other than mediating breathing‐related contractions, such as adjusting diaphragm activity during postural alterations or vomiting.
Mapping of multisynaptic inputs from the midbrain, diencephalon, and cerebral cortex to phrenic motoneurons using transneuronal transport of rabies virus N2C in the cat
Authors: Lois James H , Rice Cory D , Mehta Ajeet S , Mehta Amar S , Kalash Ronny , Yates Bill J
Published: June 2021

The transneuronal tracer rabies virus N2C can be utilized to the multisynaptic inputs controlling the activity of a muscle, since infected neurons do not quickly exhibit lysis or pathology. In the present study, we injected rabies virus N2C into the diaphragm of cats in order to map the locations of neurons in the midbrain, diencephalon, and cerebral cortex that participate in regulating breathing. Labeled cells in these regions were not evident until after a large number of brainstem neurons were infected, suggesting that the higher centers regulate diaphragm activity indirectly through connections with bulbospinal pathways. The earliest‐infected neurons in the midbrain were located in the nucleus coeruleus, red nucleus, periaqueductal gray, Edinger‐Westphal nucleus, and tegmental fields. When infection became more advanced, additional midbrain areas also exhibited labeling, including the cuneiform nucleus, raphe nuclei, inferior colliculus, and substantia nigra. The earliest‐infected diencephalon neurons were observed in the dorsal and lateral hypothalamic nuclei, although animals with advanced infections also exhibited labeling in a number of hypothalamic areas, the ventral tegmental area, zona incerta, fields of Forel, and a variety of other regions. The first‐infected cortical neurons were located anterior and posterior to the cruciate sulcus (motor cortex), as well as in prefrontal cortical areas. These data suggest that a complex, distributed network of neurons indirectly contributes to controlling diaphragm contractions.
Neonatal intermittent hypoxia induces sex‐specific enhancement of hypoxic ventilatory response in rat pups
Authors: Julien Cecile , Niane Lalah , Bairam Aida , Joseph Vincent
Published: June 2021

We tested the hypothesis that chronic intermittent hypoxia induces gender‐specific effects on hypoxic ventilatory response (HVR) in newborn rats. Rat pups were exposed with their dams to normal room air (controls) or to chronic intermittent hypoxia (IH: O2 levels cycled between 21% and 5% every 10 minutes for 1h followed by 1h under normoxia, 24h/day) during postnatal days 1–10. Minute ventilation (VE) was measured in 10‐day‐old male and female rat pups using whole‐body plethysmography. To assess HVR, VE was measured in normoxia and during acute hypoxia (5% achieved in 60 seconds), and expressed as % changes vs. baseline. IH induced a growth‐delay of similar magnitude in males and females. Under normoxia, respiratory frequency was slightly lower in both male and female IH rats vs. controls, but VE was similar. In males, HVR was higher in IH rats (+57±13%) vs. controls (+32±8%, p=0,001). In females, HVR was similar between neonatal IH (+35±13%) and control rats (+42±7%, p=ns). This study shows that neonatal IH increases HVR specifically in male rat pups, and suggests that apneas may have sex‐specific effects on respiratory control in neonates. (Founded by FRSQ, Sick kids foundation/CIHR).
Neural control of cutaneous vasoconstriction during lower‐body negative pressure (LBNP) in the heat stressed individual
Authors: Shibasaki Manabu , Rasmussen Peter , Secher Niels H. , Crandall Craig G.
Published: June 2021

The decrease in cutaneous vascular conductance (CVC) during LBNP in the heat stressed human could be mediated by neural and non‐neural mechanisms. This project further investigated this question. Nerve activity to a forearm was blocked (via axial blockade) in seven healthy subjects. Two intradermal microdialysis probes were placed within the skin of the blocked area. Forearm skin blood flow was measured by laser‐Doppler flowmetry over these microdialysis probes, as well as from skin of the contralateral (non‐blocked) forearm. The absence of sweating and cutaneous vasodilation during heat stress confirmed an effective block. Upon this confirmation, adenosine was perfused through one microdialysis probe to increase skin blood flow similar to that of the non‐blocked site. After internal temperature was elevated ~1.0 °C, 30 mmHg LBNP was applied for 10 min, or until the onset of presyncopal symptoms. LBNP significantly decreased CVC at all three sites (unblocked site: −6.1±2.7; blocked site: −2.5±1.3%max; blocked site with adenosine: −5.4±2.2%max), although the magnitude of reduction in CVC was not different between unblocked and adenosine sites. When combined with prior data, these findings suggest that reductions in CVC to LBNP of heat stressed individuals could have both neural and non‐neural components.

Study funded by part by NIH HL61388 & HL84072
The influence of exercise‐induced dehydration on thermal sensation and comfort
Authors: Nagashima Kei , Tokizawa Ken , Yasuhara Saki , Nakamura Mayumi , Crawshaw Larry
Published: June 2021

[Introduction] Dehydration attenuates autonomic thermoregulation in the heat such as sweating and skin blood flow in human. Hyperosmolality in the extracellular fluid is thought to be involved in this mechanism. In contrast, behavioral thermoregulation (i.e. heat escape behavior) is augmented after hypertonic‐saline injection in rats (Nagashima et al., Am J Physiol 2001). However, it remains unknown if human activate behavioral thermoregulation during dehydration. [Methods] Ten healthy young men participated in the present study. Body and skin temperature were monitored. Rating for thermal sensation and comfort were conducted. After ~45 min rest at 27°C, they did 50‐min ergometer exercise, which was at the level of 40% peak VO2 or ~15% peak VO2 at an ambient temperature of 35°C or ~24°C, respectively. Subjects returned to the rest at 27°C until body and skin temperature being restored. Ambient temperature was then changed from 22°C to 38°C. [Results] Body weight decreased by 1.1 ± 0.1% after the exercise at 35°C and 0.2 ± 0.1% at ~24°C. Plasma osmolality increased by 6 ± 1 mosmol/kg•H2O at 35°C, but did not change at ~24°C. Thermal sensation and comfort in the heat were augmented (i.e. feeling hotter and more uncomfortable) after the exercise at 35°C. [Conclusion] Dehydration increases thermal sensation and comfort in the heat in human, which are closely related to behavioral thermoregulation.
The effect of passive exercise on post‐exercise autonomic cardiovascular control
Authors: Volianitis Stefanos , Vorluni Luigi
Published: June 2021

Following dynamic exercise a blood pressure (BP) reduction compared to pre‐exercise values (post‐exercise hypotension, PEH) has been reported. One of the suggested mechanisms responsible for PEH is the vasodilatating effect of exercise‐born substances that may alter the arterial baroreflex function. To evaluate the contribution of exercise‐induced vasodilatating substances on post‐exercise autonomic cardiovascular control we measured BP and pulse interval (PI) continuously and noninvasively in 14 subjects pre‐ and post 20 min of passive cycling at 75 rpm, where the production of vasodilatating substances is considered absent The arterial baroreflex was evaluated with the cross‐correlation method (xBRS) for the computation of time‐domain baroreflex sensitivity on spontaneous BP and PI variability. xBRS computes the greatest positive correlation between beat‐to‐beat systolic BP and PI, and when significant at P = 0.01, slope and delay are recorded as one xBRS value. Post‐exercise, systolic and diastolic BP were elevated compared to rest (126 ± 7 vs 119 ± 8 and 76 ± 6 vs 72 ± 5 mmHg, P < 0.05, respectively). There was no change in xBRS post‐exercise compared to rest (14.1 ± 8.2 to 14.4 ± 8.3 ms/mmHg, respectively). In conclusion, these findings suggest that the vasodilatating effect of exercise‐born substances is critical for the development of PEH.
Ascorbic acid does not influence skeletal muscle contraction‐induced rapid vasodilation in older healthy humans
Authors: Simpson Carrie B , Kirby Brett S , Carlson Rick E , Schrage William G , Voyles Wyatt F , Dinenno Frank A
Published: June 2021

Age‐related increases in oxidative stress impair endothelium‐dependent vasodilation in humans, which could possibly explain our recent observations of blunted contraction‐induced rapid vasodilation in older adults. Therefore, we tested the hypothesis that endothelial dysfunction impairs the rapid vasodilator responses to brief muscle contractions in aging humans. In 13 young (24±1 yrs) and 13 healthy older adults (62±2 yrs), we measured forearm blood flow (FBF; Doppler ultrasound) and calculated vascular conductance (FVC) responses to single, 1 sec dynamic contractions at 10, 20, and 40% maximum voluntary contraction (MVC) before and after intra‐arterial administration of ascorbic acid (AA). Prior to AA, peak vasodilator responses to all contraction intensities were impaired ~35–50% in older adults (P<0.05), as were the immediate (1st cardiac cycle post contraction) vasodilator responses at 20 and 40% MVC (~50%; P<0.05). AA did not influence vasodilator responses to sodium nitroprusside in either group, but significantly improved vasodilation to acetylcholine in older adults only (P<0.05). In contrast, AA did not influence contraction‐induced rapid vasodilation in either group (all NS). Together, our findings suggest that endothelial dysfunction is not the primary mechanism underlying impaired contraction‐induced rapid vasodilation with human aging.

Supported by AG‐027150
Leucine‐enriched essential amino acid and carbohydrate ingestion in women increases skeletal muscle mTOR and Akt/AS160 signaling
Authors: Drummond Micah J , Dreyer Hans C , Fujita Satoshi , Volpi Elena , Rasmussen Blake B
Published: June 2021

Men respond to an essential amino acid and carbohydrate ingestion with a rapid and large increase in mTOR signaling and muscle protein synthesis. However, it is not known whether women have a similar response. We hypothesized that anabolic signaling and changes in cell energy status would be similar between men and women following nutrient ingestion. Seven young female subjects ingested a leucine‐enriched essential amino acid (0.35g/kg fat free mass) and carbohydrate (0.5 g/kg fat free mass) nutrient solution. Muscle biopsies from the vastus lateralis were obtained at baseline and 1h following nutrient ingestion. Immunobloting techniques were used to identify the phosphorylation pattern of three key nutrient‐sensitive signaling pathways: mTOR, Akt/AS160 and AMPK. mTOR signaling was enhanced as indicated by significantly increased phosphorylation of mTOR at Ser2448 and its downstream effectors S6K1 (Thr389) and 4E‐BP1 (Thr37/46) while eEF2 phosphorylation at Thr56 was significantly decreased (P<0.05). In addition, Akt (Thr473) and AS160 phosphorylation were increased; however, AMPKα phosphorylation at Thr172 was unchanged. We conclude that mTOR signaling in women following essential amino acid and carbohydrate ingestion is similar to the response in men. Furthermore, the increase in glucose uptake following nutrient ingestion is associated with an increased phosphorylation of human muscle AS160.

Funded by NIH grant # R01 AR049877
Muscle Atrophy and Increased Atrogin‐1 Occur in Fast‐ but not Slow‐Muscle Early in Acute Streptozotocin‐Induced Diabetes
Authors: Templeton Erika L , Motyl Katie , McCabe Laura , Wiseman Robert
Published: June 2021

The onset of diabetes is characterized by a loss of glucose homeostasis as well as a loss in lean body mass. Recent reports suggest that muscle loss may be fiber type specific and correlated with oxidative capacity (Sandri et al., 2006. PNAS, 103:16260–5). To test this hypothesis a streptozotocin (STZ) ‐treated animal model was used. BALB/c mice were injected with 40 ug/g STZ over the course of 5 days with sham injected controls. Maximal weight loss was observed 5 days post‐injection. Animals were sacrificed at this time and plasma, tibialis anterior (TA) (fast‐twitch), and soleus (SOL) (slow‐twitch), muscles were harvested, weighed and rapidly frozen at −80°C. Plasma glucose levels rose from approximately 150 to 270 mg/dL by day 5. TA muscles decreased in mass from 46 ± 2 to 39 ± 1 mg with no detectable change in SOL weights. Real‐time PCR showed atrogin‐1 mRNA was two‐fold increased in STZ‐treated TA muscles relative to sham‐injected controls but there was no change in SOL expression. Both muscle mass and atrogin‐1 expression show fast‐fiber specific effects under STZ‐treatment that are absent in the more oxidative SOL muscles. These data suggest that oxidative capacity and/or genes controlling oxidative capacity protect against the loss in mass possibly by retarding atrogin‐1 expression.

Supported by NIH DK 061184
Building centromeric chromatin
Authors: Gerton Jennifer Lynn , Camahort Raymond , Mattingly Mark , Shivaraju Manjunatha , Li Bing , Florens Laurence , McNairn Adrian
Published: June 2021

We are interested in understanding how centromeres are specified and how centromeric chromatin forms. To this end, we immunopurified the centromeric histone H3 variant Cse4 from budding yeast and subjected this preparation to MudPIT analysis to identify interacting proteins. Peptides of Scm3 were identified. Scm3 is essential for cell viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint, being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin following DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. This research is supported by the Stowers Institute for Medical Research.
Chronic hypoxia alters anoxia‐induced activation of ATP‐sensitive K+ (KATP) channels in second‐order peripheral chemoreceptor neurons in the NTS
Authors: Zhang Weirong , Mifflin Steve
Published: June 2021

The impact of tissue hypoxia on 2nd‐order NTS neurons receiving chemoreceptor afferent inputs is unknown. We investigated the neuronal response to anoxia (bath solution bubbled in 95% N2/5% CO2 vs control 95% O2/5% CO2) in normoxic rats (NORM) and rats exposed to chronic hypoxia (CH; 7‐10 days at 10% FIO2). Whole‐cell recordings of NTS 2nd‐order neurons identified following DiA labeling of the carotid bodies were obtained in a brainstem slice. In 8 cells from NORM rats, anoxia (3 min) induced outward currents of 13.1±1.0 pA that reversed at −73±2 mV and decreased input resistance (59±2%). This current was completely blocked by the KATP channel blocker tolbutamide (100 μM). In NORM rats the KATP channel opener diazoxide (200 μM, 3 min) evoked an outward current of 27.7±5.5 pA (n=4), which was blocked by tolbutamide. In 10 CH neurons the same anoxia protocol induced a significantly smaller outward current (5.9±1.5 pA, p<0.01) and decreased input resistance (21±2%, p<0.01). In CH, diazoxide (200 μM, 3 min) induced an outward current significantly smaller than in NORM neurons (2.5±0.6 pA, n=4, p<0.01). These results suggest that anoxia activates KATP channels in 2nd‐order NTS neurons receiving chemoreceptor afferent inputs. CH reduces KATP channel function in NTS neurons. This could represent a neuronal adaptation to CH that preserves neuronal excitability in these crucial relay neurons in peripheral chemoreflex pathways.
Intermittent Hypoxic Conditioning Suppresses Angina And Arrhythmias In Patients With Ischemic Heart Disease
Authors: Manukhina Eugenia , Lyamina Svetlana , Mallet Robert T , Downey H Fred , Lyamina Nadezhda
Published: June 2021

Intermittent hypoxic conditioning (IHC) greatly reduces ischemia‐reperfusion cardiac injury and arrhythmias in animal experiments. The present study evaluated clinical benefits of IHC in 53 patients with ischemic heart disease (IHD) and extrasystolic arrhythmias. Twenty daily sessions of IHC were performed by alternately inspiring 10% O2 and atmospheric air for 3 min each (each hypoxic exposure 20–60 min/session). Twelve patients received sham IHT (inspiration of atmospheric air; SHAM). Thirteen patients received only drug therapy (CONT). ECG was monitored for 24 h before and after 20 days of respective therapy and at 3 months after IHC. All patients received standard medical therapy for IHD. Table shows % of patients with complete (CS) or partial (PS) suppression of arrhythmias and change in anti‐anginal medication (Δ Dose). Percent reduction in frequency of anginal attacks (#AA) and % reduction in AA duration are also reported.

IHC produced no serious adverse effects, although initially IHC caused mild dizziness in 12% of patients and drowsiness in 31% of patients. Three months after IHC therapy, 88% of patients had no increase in arrhythmias or AA. Therefore IHC safely augments standard medical therapy for patients with IHD.
Post‐translational modification of peptidylglycine α‐amidating monooxygenase by intermittent hypoxia
Authors: Kumar Ganesh K , Prabhakar Nanduri R
Published: June 2021

Peptidylglycine α‐amidating monooxygenase (PAM) catalyzes the conversion of glycine‐extended peptide precursors to biologically active α‐amidated peptides in a copper, ascorbate and molecular oxygen‐dependent manner. In this study, we investigated the effects of chronic intermittent (CIH) and sustained hypoxia (CSH) on PAM activity and expression in the rat brainstem. CIH but not comparable duration of CSH increased PAM activity (by ∼ 3‐fold) resulting in higher Vmax. CIH enhanced ascorbate‐, but not Cu2+‐dependent enzyme activity. Western blot analyses showed that CIH increases the formation of low molecular weight with a concomitant decrease in high molecular weight PAM‐related proteins. CIH increased trypsin‐like activity (∼2‐fold) in the brainstem. Trypsin activated PAM in the control but not in CIH brainstem extracts. CIH increased ROS in the brainstem. Anti‐oxidants abolished CIH‐induced increases in PAM and trypsin‐like activities as well as proteolytic processing of PAM. Taken together, these observations suggest that CIH activates PAM in the rat brainstem by mechanism(s) involving ROS‐dependent post‐translational proteolytic processing of the enzyme (supported by NIH HL‐25830).
Phospholamban expression in mouse soleus muscle is downregulated after chronic running wheel exercise
Authors: de Snoo Maartje , Everts Maria , Joosten Brian , Doornenbal Arie , Schot Arend , Jeneson Jeroen
Published: June 2021

Endurance exercise is typically associated with a shift in myosin heavy chain (MyHC) towards slower phenotype and/or an increase in mitochondrial density in skeletal muscle. Wheel running exercise has been extensively used to study these processes in mice in vivo. However, results concerning the ‘classic’ endurance‐type fast‐to‐slow fiber transition are ambiguous. We hypothesized that the specific running pattern of the mouse–i.e. short bouts at high speed‐ underlies this phenomenon and that subsequent adaptation processes involve Ca2+ handling mechanisms. Soleus muscles of male C57Bl/6 mice were isolated after six weeks of voluntary wheel running and characterized physiologically. Trained muscles had improved fatigue resistance, improved capacity for relaxation during serial electrical stimulation, and consumed 1.3‐fold more oxygen than control muscles. Biochemical analysis at the protein level revealed that neither MyHC composition nor sarcoplasmic reticular Ca2+ ATPase (SERCA) content had changed during training. Quantitative RT‐PCR revealed that phospholamban (PLB) mRNA content was 60% lower than control, while SERCA mRNA content was unchanged. This study showed that chronic wheel running in mice improves fatigue resistance of soleus muscle via an improved capacity for relaxation through downregulation of PLB expression resulting in disinhibition of SERCA.
Redox regulation of diaphragm proteolysis during mechanical ventilation
Authors: McClung Joseph M , Whidden Melissa A , Kavazis Andreas N , Falk Darin J , DeRuisseau Keith C , Powers Scott K
Published: June 2021

Prevention of oxidative stress via antioxidants attenuates mechanical ventilation (MV) diaphragm myofiber atrophy. The specific redox sensitive mechanisms for this attenuation remain unknown. We hypothesized that skeletal muscle proteolytic activity is a potential site of diaphragm redox regulation during MV. Rats were assigned to: 1) control (Con): 2) 6‐hours of MV (6hr MV); 3) 6‐hours of MV with the antioxidant, Trolox (6hr MVT), 4) 18‐hours of MV (18hr MV); and 5) 18‐hours MV with Trolox (18hr MVT). Trolox did not attenuate MV induced increases in ubiquitin‐protein conjugation, poly‐ubiquitin and proteasomal subunits (20S proteasome α‐subunit 7, 14kDa‐E2, and proteasome‐activating complex PA28) gene expression. However, antioxidant administration reduced both chymotrypsin‐like and PGPH‐like 20‐S proteasome activities. Trolox attenuated MV‐induced increases in the percentage of easily releaseable diaphragm myofilament protein. These data suggest that the protective effect of antioxidant administration on the diaphragm during MV occurs primarily by decreasing myofilament protein substrate availability to the proteasome. This work supported by National Institutes of Health (R01 HL072789).
Exogenous ATP regulates excitation‐induced force in isolated muscle through the P2Y receptor family
Authors: Broch‐Lips Martin , Pedersen Thomas Holm , Nielsen Ole Bækgaard
Published: June 2021

In skeletal muscles, contractions elicit a release of ATP into the extracellular space. Extracellular ATP has been recognized as an autocrine and paracrine signaling molecule acting on two purinergic receptor families; the metabotrophic P2Y receptors and the ionotropic P2X receptors. In this study we investigate the role of P2 receptors on the excitability and contractility of isolated skeletal muscle.

Tetanic force production and M‐wave amplitude and area were investigated in isolated rat soleus muscles incubated in Krebs‐Ringer buffer. Exogenously added P2‐receptor agonist (ATP) and phospholipase C (PLC) inhibitor (U73122) were used to investigate P2‐receptor mediated effects on muscle function under conditions with compromised muscle excitability.

When the excitability of muscles was depressed by elevating [K+]o to 10 mM, tetanic force was reduced to 24 ± 2 % of initial force at 4 mM K+. However, upon addition of 1 mM ATP, force was recovered to 65 ± 8 % of initial force at 4 mM K+ (P < 0.001, n = 5). The force recovery was accompanied by a recovery of M‐wave area and amplitude from 20 ± 5 to 81 ± 11 % (P < 0.05) and 8 ± 1 to 47 ± 11 % (P < 0.05) of initial M‐wave area and amplitude at 4 mM K+, respectively (n = 4). ATP induced force recovery could be eliminated by 50 μM PLC inhibitor indicating that the force recovery was mediated by the P2Y receptor family.

Danish Medical Research Council (j. nr. 271‐05‐0304)
Differential Protein Expression in Heart in UT‐B Null Mice with Cardiac Conduction Defects
Authors: hao Yu , Meng yan , Zhao li‐juan , Wang li‐shun , Jin xian , Li yang , Chen guo‐qiang , Zhao xue‐jian , Yang bao‐xue
Published: June 2021

Cardiac conduction defects were found in transgenic mice deficient in UT‐B. To investigate the molecular mechanisms of the conduction defects caused by UT‐B deletion, we studied the protein expression profiles of heart tissue in UT‐B null mice vs age‐matched wild‐type mice at different developmental stages. Using two‐dimensional electrophoresis in combination with matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry, we found that 26 proteins were modulated in the myocardium of UT‐B null mice. Of the modulated proteins, troponin T, cardiac (TNNT2) was up‐regulated 5.31 ± 0.45 (n = 3) folds in 16 weeks old UT‐B null mice and down‐regulated 3.99 ± 0.40 folds in 52 weeks old mice. Atrial natriuretic peptide (ANP) was up‐regulated 4.18 ± 0.61 folds in 52 weeks old UT‐B null mice with AV block. The modulation of these 2 proteins was confirmed by the Western‐blot analysis. The results indicate that UT‐B deletion caused the expression regulation of TNNT2 and ANP in mouse heart with cardiac conduction defect, which provides new clues to the mechanism involved in cardiac conduction defect.
The N‐terminus of myosin binding protein‐C diminishes the Ca2+ dependence of the rate of tension redevelopment in permeabilized cardiac trabeculae
Authors: Harris Samantha , Razumova Maria V , Bezold Kristina L , Tu An‐yue , Regnier Michael
Published: June 2021

Myosin binding protein‐C (MyBP‐C) is a thick‐filament protein that limits cross‐bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP‐C affects contraction we assessed effects of recombinant N‐terminal domains of cardiac (c) MyBP‐C on contractile properties of permeabilized rat cardiac trabeculae. Here we show that N‐terminal fragments of cMyBP‐C containing the first two immunoglobulin (Ig) domains of cMyBP‐C (i.e., C0 and C1) plus the unique linker sequence termed the MyBP‐C “motif” increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (i.e., ktr) at sub‐maximal levels of Ca2+. Rates of tension redevelopment were accelerated to near maximal even at the lowest levels of force generation, thus significantly diminishing the Ca2+‐dependence of ktr. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca2+ (pCa 9.0) and inhibited maximum Ca2+‐activated force at pCa 4.5. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP‐C that cooperatively activates the thin filament. This work supported by NIH HL080367 (to SPH) and HL 061683 (to MR).
Activation of Store‐Operated Calcium Entry Channels Stably Increases Membrane‐Localized Calcium
Authors: Barry Christina Jean , Cioffi Donna L. , Stevens Troy
Published: June 2021

Inflammatory agonists deplete calcium from the endoplasmic reticulum and activate calcium entry in a process referred to as store‐operated calcium (SOC) entry. Calcium that permeates through SOC channels is sufficient to increase permeability although it is not clear why calcium entry, and not calcium release, is necessary to increase permeability. In our present studies, we tested the hypothesis that calcium entry is necessary to produce a sustained increase in intracellular calcium that is localized at the cell membrane. To test this hypothesis, pulmonary artery endothelial cells were loaded with either a cytosol‐localized calcium fluorophore (Fura‐2AM) or a membrane‐localized fluorophore (FFP‐18). Membrane and cytosolic calcium concentrations were calculated using the Grynkiewicz equation. Baseline membrane calcium concentrations were higher than cytosolic calcium concentrations, indicating a calcium gradient from the plasma membrane to the bulk cytosol. Upon thapsigargin‐induced SOC entry, both cytosolic and membrane calcium increased. However, cytosolic calcium decayed over time whereas membrane calcium was sustained. Collectively, these observations indicate that calcium entry through SOC channels is necessary to stably increase membrane‐associated calcium, likely necessary to induce the inter‐endothelial cell gaps that precede edema formation. Supported by HL60024.
Dynamic motion of paxillin on actin filaments in living endothelial responses to shear stress
Authors: Hu Ying‐Li , Chien Shu
Published: June 2021

Paxillin, an adaptor protein that is associated with the cytoplasmic domains of integrins and localized at focal adhesions (FAs), interacts with cytoskeletal and signaling proteins to regulate the assembly, disassembly, and signaling of FAs. In living endothelial cells (ECs), fluorescence imaging of the intracellular dynamics of paxillin and actin filaments indicated that actin filaments are important in paxillin assembly at FAs. Using a laser scanning confocal microscopic imaging system, three‐dimensional (3‐D) images were obtained from a z‐series collection of dual‐color optical sections of fixed cells. The results showed the co‐localization of paxillin on actin filaments as fibrous structures, as well as clusters. In living ECs under flow condition, concurrent monitoring of the intracellular dynamics of DsRed2‐paxillin and GFP‐actin by time‐lapse video recording and dual‐color fluorescence imaging showed that the paxillin fibrous structures in ECs moved in close association with and along the actin filaments. Our findings suggest that the actin network not only plays an important role in the assembly/disassembly of paxillin at FAs, but also serves as a track for the intracellular transport of paxillin, which plays an important role in cellular signaling cascades. This work was supported by National Heart, Lung, and Blood Institute Research Grants HL‐064382, HL‐080518, and HL‐085159 (S.C.).
IL‐4 induces interleukin‐6 (IL‐6) expression in human aortic endothelial cells
Authors: Lee Yong Woo , Lee Won Hee
Published: June 2021

Previous studies have demonstrated that the pro‐oxidative and pro‐inflammatory pathways within vascular endothelium play an important role in the initiation and progression of atherosclerosis. The present study was designed to examine the hypothesis that interleukin‐4 (IL‐4) may up‐regulate expression of pro‐inflammatory cytokine IL‐6 in human aortic endothelial cells (HAEC). Quantitative real‐time RT‐PCR and enzyme‐linked immunosorbent assay (ELISA) showed that exposure of HAEC to IL‐4 significantly up‐regulated the mRNA and protein expression of IL‐6. Overexpression of IL‐6 was further confirmed in aorta and serum isolated from IL‐4‐injected mice. In addition, pretreatment with antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and epigallocatechin gallate (EGCG), significantly and dose‐dependently inhibited IL‐4‐induced IL‐6 expression in HAEC. These results suggest that IL‐4 can induce IL‐6 expression through antioxidant‐sensitive mechanisms in human aortic endothelial cells. These data may contribute to understanding the cellular and molecular mechanisms involved in IL‐4‐mediated progression of atherosclerosis and development of therapeutic strategies for the prevention of atherosclerotic lesion development specifically targeted against inflammatory pathways. (This work was supported by Grant Number R15HL085229 from the National Heart, Lung, and Blood Institute)
PAF and H2O2 act synergistically to increase endothelial cell oxidant stress
Authors: Wang Zhanfei , Bian Zhouyan , Rui Tao , Kvietys Peter R.
Published: June 2021

We have previously shown that alveolar macrophages from septic mice can 1) induce an oxidant stress in pulmonary endothelial cells (PEC) and 2) promote neutrophil (PMN) transendothelial migration. Both of these responses can be blocked by catalase or a PAF receptor antagonist. In the present study, we assessed whether PAF and/or H2O2 can induce an oxidant stress in endothelial cells. PEC were isolated from murine lungs and exposed to different concentrations of H2O2 (25 – 200 μM) and PAF (10‐10 – 10‐7 M) for 1 hr. Subsequently, PEC oxidant stress was assessed using DHR 123 oxidation as an index. H2O2 increased PEC oxidant stress only when used at the highest concentration (200 μM), while PAF did not increase PEC oxidant stress at any of the concentrations tested. When PAF and H2O2 were added simultaneously to PEC there was a potentiation of PEC oxidant stress. Our findings indicate that PAF and H2O2 act synergistically to promote endothelial cell oxidant stress, most likely through the activation of NADPH oxidase.
Sphingosine 1‐phosphate induces platelet/endothelial cell adhesion molecule‐1 phosphorylation in human endothelial cells through cSrc and Fyn
Authors: Huang Yu‐Ting , Lee Hsinyu
Published: June 2021

Sphingosine 1‐phosphate (S1P) is a multifunctional lysophospholipid. Through binding with a specific family of G protein‐coupled receptors, S1P activates various cellular functions in endothelial cells. Platelet/endothelial cell adhesion molecule‐1 (PECAM‐1) is a 130‐kDa transmembrane protein expressed predominantly on the surface of platelets and endothelial cells. Upon activation, the cytoplasmic tyrosine residues could be phosphorylated and regulate several cellular functions, including endothelial integrin activation and diapedesis. In this study, we first demonstrated that S1P induced PECAM‐1 tyrosine phosphorylation in human umbilical cord vein cells (HUVEC) by immunoprecipitation and western blot. The induction was blocked by pertussis toxin and PP2, the inhibitor for Gi and Src family kinases (SFK). In addition, S1P‐induced SFK activation at endothelial cell borders was also observed by western blot and confocal microscopy. Two members of SFK, cSrc and Fyn, were dominantly expressed in HUVEC and targeted with specific siRNA. The induction effect of S1P on PECAM‐1 tyrosine phosphorylation was significantly suppressed in HUVEC cotrasfected with both siRNA. These results would help to elucidate the regulatory mechanism of S1P in PECAM‐1 functions in human endothelial cells.
Mechanism of myosin phosphatase inhibition via phosphorylation of MYPT1 subunit by RhoA/ROCK
Authors: Eto Masumi , Choudhury Nandini , Stevenson Andra , Somlyo Avril V
Published: June 2021

Smooth muscle tone is determined by reciprocal activities of myosin light chain kinase and phosphatase in response to agonist stimulation and nitric oxide production. Myosin phosphatase, a heterotrimeric form of protein phosphatase‐1, is inhibited upon activation of RhoA/ROCK, and the inhibition is cancelled in response to cyclic nucleotide signal. MYPT1, a regulatory subunit of myosin phosphatase, is phosphorylated at Thr696 and 853 during agonist‐induced contraction. Here, we investigated mechanisms underlying the inhibition of myosin phosphatase through phosphorylation of MYPT1, using GST‐fusion peptides of MYPT1 segments including phosphorylation sites. GST‐MYPT1(654‐880) peptide including both Thr696 and 853 and shorter peptides including either phosphorylation site, 654‐714 with Thr696 and 697‐880 with Thr853, induced the contraction of beta‐escin permeabilized rabbit ileum at constant Ca2+ of pCa 6.3. The MYPT1 peptide‐induced force completely declined by addition of ROCK inhibitors, as well as addition of 8Br‐cGMP. Purified myosin phosphatase was inhibited by phosphorylated GST‐MYPT1(654‐714) and (696‐880) peptides, with IC50 of 1 nM and 20 nM, respectively. These peptides directly docked at the active site of myosin phosphatase catalytic subunit. Thus, phosphorylation of MYPT1 at Thr696 and 853 converts the region into autoinhibitory domain of myosin phosphatase that mediates agonist stimulation into smooth muscle contraction.
Decreased frequency of transient outward BK currents in cerebral myocytes following subarachnoid hemorrhage
Authors: Koide Masayo , Krishnamoorthy Gayathri , O’Connor Kevin P , Nelson Mark T , Wellman George C
Published: June 2021

Cerebral vasospasm is a prolonged vasoconstriction causing substantial morbidity and mortality in patients following aneurysmal and subarachnoid hemorrhage (SAH). The mechanisms underlying this pathology remain unclear, but may involve decreased K+ channel activity in cerebral artery myocytes. Ca2+ sparks are localized intracellular Ca2+ release events leading to the transient activation of large conductance Ca2+‐activated K+ (BK) channels. Here, we evaluated transient BK currents in freshly isolated cerebral artery myocytes obtained from a rabbit model of SAH. Transient whole cell BK currents and single channel BK properties were examined using the perforated whole cell and single channel patch‐clamp technique, respectively. We also examined BK channel α and β1 subunit expression using RT‐PCR. Transient BK current frequency was decreased in cerebral artery myocytes from SAH rabbits, however BK channel properties (voltage‐ and Ca2+‐dependence) and expression were unchanged. These data suggest decreased Ca2+ spark frequency or an uncoupling between Ca2+ spark and BK channel activation may underlie the observed decrease in transient BK frequency. A reduction in BK channel activity in cerebral artery myocytes may contribute to cerebral vasospasm following SAH. This work was supported by the Totman Medical Research Trust, the Peter Martin Brain Aneurysm Endowment, the AHA and the NIH.
NADPH oxidase derived superoxide (O2·−) upregulates intermediate‐conductance Ca2+‐activated K+ channel (KCa3.1) expression in vascular smooth muscle cells (VSMCs)
Authors: Gole Hope KA , Bowles Douglas K
Published: June 2021

Reactive oxygen species (ROS) in VSMCs, predominantly from NADPH oxidases, contribute to hypertension, atherosclerosis, and hypertrophy. Excess ROS cause oxidative stress and mediate VSMC phenotypic modulation including hypertrophy, cell migration and cell proliferation. We have previously shown that functional upregulation of KCa3.1 is required for VSMC phenotypic modulation and migration, but the effect of ROS on KCa3.1 regulation is unknown. Our objective was to determine if NADPH oxidase derived O2·− upregulates KCa3.1 expression. Cultured rat aortic SMCs were serum starved four days, drug treated 24 hours, and RNA isolated for quantitative RT‐PCR (qRT‐PCR). Basic fibroblast growth factor (bFGF, 25ng/mL) and angiotensin II (AngII, 100nM) increased KCa3.1 mRNA levels 6.0 and 3.5 fold respectively; while bFGF decreased smooth muscle myosin heavy chain (SMMHC) mRNA levels 80% (P< 0.05). NADPH oxidase inhibitors apocynin (Apo, 1mM) or diphenylene iodonium (DPI, 10uM), and superoxide dismutase (SOD) mimetic Tempol (1mM) completely inhibited KCa3.1 upregulation while having no affect on SMMHC downregulation. The inhibition resulting from Tempol was similar to Apo or DPI, indicating NADPH oxidase was the primary source of O2·− regulating KCa3.1 expression. Our findings provide a novel mechanism by which KCa3.1 expression is increased by NADPH oxidase derived O2·−. Supported by NIH RR18276, HL52490.