bluuebunny/crossref_metadata_2025 huggingface dataset:
Ossabaw miniature swine models with mutant vs. non‐mutant AMP kinase alleles for study of electrocardiographic properties during myocardial ischemia
Authors: Chawla Aarti , Spencer Stephen , McKenney Mikaela L , Schultz Kyle A , Byrd James , Alloosh Mouhamad , Mather Kieren , Sturek Michael
Published: June 2021

Myocardial ischemia activates AMP kinase (AMPK), which modulates electrophysiology. A spontaneous point mutation from Val199→Ile in the AMPK γ3 subunit of Ossabaw swine impairs AMPK function. We hypothesized that ischemia would elicit greater electrocardiographic ST segment elevation (STE) in AMPK mutant compared to non‐mutant swine. Ischemia induced by balloon occlusion of the circumflex artery was verified by angiography. AMPK mutants showed significant STE within 3 min of ischemia, while non‐mutants showed an STE of ~2mm during 15 min of ischemia. Percent survival to the STE criterion was greater in non‐mutants vs. mutants (p<0.05). Intracoronary infusion of the AMPK activator AICAR (0.5 mM) for 10 min before coronary occlusion reduced profound STE in AMPK mutants, but completely prevented STE in non‐mutants. Percent survival to the STE criterion again was greater in non‐mutants vs. mutants during ischemia. Compound C, an inhibitor of AMPK, trended to decrease time to reach STE criterion compared to AICAR. Metabolic syndrome induced by hypercaloric atherogenic diet was similar in mutants and non‐mutants. Mutants reached the STE criterion in 4 min, but the non‐mutants did not in the 15 min occlusion, thus showing a striking cardiac phenotype. In conclusion, a functional cardiac AMPK and pharmacological AMPK activation decrease ischemia‐induced ST segment elevation. (Support: NIH HL062552)
Differential Cardioprotection by Ischemic Preconditioning Mediated by Rat Chromosome 6 Is Nitric Oxide‐Dependent
Authors: Stowe David , Nabor Darren , Chen Qunli , Lazar Jozef , Riess Matthias
Published: June 2021

Brown Norway (BN) rat hearts are more resistant to ischemia‐reperfusion (IR) injury than are Dahl Salt Sensitive (SS) hearts. Moreover, IR injury is attenuated by ischemic preconditioning (IPC) in BN, but not in SS hearts. We hypothesized that the positive effect of IPC is due to genetic information on BN chromosome 6 that affects endogenous nitric oxide (NO•) production. To test this we isolated hearts from BN, SS and consomic SS6BN rats and preconditioned hearts with two 5‐min IR periods before 30 min global ischemia and 2 h reperfusion; controls (Con) had no IPC. In some hearts, NO• synthase was inhibited during IPC by L‐NAME. We monitored LV pressure and its derivatives before, during and after ischemia and measured infarct size (IS). We found that functional return on reperfusion was lower, and IS larger in SS than in BN or SS6BN Con hearts. IPC improved LV function and reduced IS in BN and SS6BN, but not in SS hearts. L‐NAME abolished all IPC‐mediated protection in BN and SS6BN hearts. Because SS6BN hearts were protected similar to BN hearts, we conclude that genetic information on BN chromosome 6 plays a key role for IPC to be effective. Also because IPC was prevented by L‐NAME, we suggest NOS and/or an upstream regulator are candidate genes for the observed genome‐dependent differences in cardioprotection in this consomic rat model. Supported by Society of Cardiovascular Anesthesiologists and Department of Veterans Affairs.
Estrous cycle phase does not influence myocardial infarction but may alter arrhythmogenicity in isolated rat hearts
Authors: Frasier Chad R , Rosenbaum Matthew D , Sloan Ruben C , Hayes Brian , Brown David A
Published: June 2021

The cardioprotective effects of estrogen are well documented in humans and animals. Although gain/loss‐of‐function studies show estrogen as a cardioprotective hormone, it is not known if physiological fluctuations during the estrous cycle influences cardiac ischemia/reperfusion (I/R) injury. We conducted this study to determine if the extent of I/R injury varies as a function of normal estrous cycling. Estrous cycle stage was determined by serum hormone levels and vaginal cytology immediately before heart excision. Isolated rat hearts underwent 25 min ischemia and 2 h reperfusion. No baseline differences were seen in heart rate, left ventricular pressure, or coronary flow. Infarct size was not affected by estrous stage (42±6, 49±4, 40±9, and 43±8% zone at risk for Proestrus (P), Estrus (E), Metestrus (M), and Diestrus (D) respectively; P>0.05). Infarct size was not correlated with serum levels of estradiol or progesterone. There was a trend for reperfusion arrhythmia fluctuations during estrous (arrhythmia scores 3.6±0.3, 4.3±0.5, 4.9±0.3, 3.3±0.6 for P, E, M, and D respectively; P=0.08). Restoration of coronary flow during reperfusion also varied with phase (P<0.05). This study demonstrates that studies using female rats have potential confounding effects of ovarian activity on cardiac I/R injury.
Anti‐arrhythmic effect of remote ischemic preconditioning is accompanied by changes in the salvage kinases in the rat heart non‐ischemic area
Authors: Simkhovich Boris Z , Dow Joan , Kloner Robert A
Published: June 2021

Remote ischemic preconditioning (RIPC) by intermittent ligation and reperfusion of femoral arteries decreases the occurrence and the duration of reperfusion arrhythmias. We sought to study whether salvage kinases (RISK) are involved in this phenomenon. Rats in the RIPC group were subjected to 3 cycles of bilateral femoral artery occlusion + reperfusion (5 min each) followed by 5 min of coronary artery occlusion with subsequent 1 min of reperfusion. In the ischemia/reperfusion (IR) group femoral arteries were exposed but not ligated. After a 30 min waiting period coronary artery was occluded and reperfused as in the RIPC group. At the end of the reperfusion the risk area was delineated with blue dye and hearts were frozen in liquid nitrogen. Non‐ischemic (NI) and ischemic (Isc) areas were separated and Western blots were performed for phospho‐Akt (p‐Akt) and phospho‐p70S6 (p‐p70S6). In the NI area the levels of kinases were reduced in the RIPC vs. IR group by 55% and 58% for p‐Akt and p‐p70S6 respectively (p<0.05). In the Isc vs. NI area of both groups kinases were equally increased and reached levels of ~2.7 baseline‐normalized units. The Isc/NI ratio in the RIPC vs. IR group was increased by 131% and 154% for p‐Akt and p‐p70S6 respectively (p<0.05). Our data suggest that RIPC increased parameters of RISK in the ischemic area to equivalent of IR alone, but reduced these parameters in the NI zone to a greater extent than IR.
Vitamin E attenuates P. aeruginosa‐mediated injury in pulmonary endothelial cells
Authors: Anjum Naseem , Iles Karen Elizabeth , Traber Maret G , Creighton Judy , Matalon Sadis , Pittet Jean-Francois
Published: June 2021

Infections caused by the opportunistic pathogen P. aeruginosa are a serious complication in critically ill patients with often lethal consequences. The high mortality of patients who develop P. aeruginosa pneumonia is associated with acute lung injury, characterized by airspace flooding. We have previously shown that in endothelial cells P. aeruginosa induces the small G‐protein RhoA, increasing actin stress fiber formation, and permeability. Herein, we hypothesize that the antioxidant vitamin, Vitamin E (Vit E) will be beneficial in preventing P. aeruginosa‐mediated damage. In rat microvascular endothelial cells (RMVEC), exposure to the PAK‐strain of P. aeruginosa (MOI 80 for 10 minutes), increased ROS production. This was completely prevented with pretreatment with 2 doses of Vit E, (18 and 1 hr prior to administration of PAK). Electric cell‐substrate impedance sensing (ECIS) was used as a functional assay to measure changes in resistance in confluent monolayers following exposure to PAK (MOI 80). PAK exposure decreased resistance indicating increased permeability. This decrease was attenuated by pretreatment with Vit E. RhoA activation and stress fiber formation by PAK was also completely blocked by pre‐treatment with Vit E. These data suggest a ROS‐component to PAK‐mediated damage and indicate a possible therapeutic role for Vit E.
Expression of T‐cadherin in a hereditary hypercholesterolemic swine model
Authors: Arce-Esquivel Arturo A. , Turk James R. , Laughlin M. Harold
Published: June 2021

Decreased adiponectin levels correlate with reduced vascular function and increased CAD. T‐cadherin is a major receptor for adiponectin in the vasculature. To evaluate the expression of Tcadherin in athero‐prone and athero‐resistant arteries of a hereditary hypercholesterolemic (HH) swine model of atherosclerosis. We tested the hypothesis that exercise training would increase the expression of T‐cadherin in atherosclerotic lesions of HH swine. Fifteen HH pigs were randomly assigned into either exercise trained (EX) or sedentary groups (SED). Exercise training consisted of a 16 week progressive treadmill training program. LAD, RCA and internal mammary artery (IM) were harvested. IHC was performed in the entire vessel wall in order to quantify the presence of T‐cadherin. The average percentage for Tcadherin staining was significantly higher in the athero‐resistant IM. Expression of T‐cadherin tended to be greater in LAD, RCA and IM from EX pigs. The differential expression of T‐cadherin between coronary and mammary arteries may in part mediate their opposite response to a pro‐atheroslcerotic stimulus, and that endurance training seems to enhance the levels of T‐cadherin of this model of disease. Supported by NIH HL52490.
Effects of Exercise Training on NASH‐Induced Oxidative Stress in Heart of Rats
Authors: Huang Wei-Chih , Chan Che-Chang , Hung Shan-Ken , Yang Ai-Lun , Tsai Shiow-Chwen
Published: June 2021

Methionine‐choline‐deficient (MCD) diet will develop Non‐alcoholic steatohepatitis (NASH) is characterized, is similar to the histopathologic changes that occur in NASH in humans. The influence of NASH on heart is still unclear.

PURPOSE

To investigate the interaction of exercise training and MCD diet on oxidative stress in heart.

METHODS

Adult male Sprague Dawley rats were randomly divided into 5 groups: control (fed normal diet, W0), MCD diet fed for 9 weeks or 14 weeks combined without (W9, W14) or with exercise training (W9+Ex, W14+Ex). The exercise training was treated for 4 weeks before sacrifice. The AST level in plasma and the antioxidation activity of liver tissue were assay by commercial kit. The statistical analysis was done using the one‐way ANOVA for independent samples.

RESULTS

MCD diet showed a rapid induction of AST and hepatic steatosis, loss of body weight and heart weight. The lipid peroxidation levels of W14 and W14+Ex were increased. Cpmpared wth W0, The activity of SOD were significantly increased in W14 (p<0.0001) and in W14+Ex (p=0.012). Compared with W14, W14+Ex decrease the activity of SOD, but increase the activity of catalase. The increased production of GSH and GSSG in W9 were due to the increased activity of GPx and GR.

CONCLUSION

Exercise training enhanced the activity of catalase and decreased the production of lipid peroxidation in LV.
Nitric oxide synthase (NOS) inhibition blunts the kinetics of vasodilation during dynamic forearm exercise
Authors: Mohamed Essa Ahmed , Joyner Michael J. , Walker Branton G. , Casey Darren P.
Published: June 2021

We tested the hypothesis that local inhibition of NOS would 1) alter the kinetics of vasodilation during dynamic forearm (FA) exercise and 2) prolong the time to reach steady state (SS). Subjects (n=22) performed rhythmic FA exercise (20% of maximum) during saline infusion (control) and NOS inhibition (L‐NMMA). Additionally, a subset of subjects performed a third trial of FA exercise during combined inhibition of either 1) NOS and adenosine (aminophylline; n=9), or 2) NOS and prostaglandins (Ketorolac; n=8). Forearm vascular conductance (FVC; ml/min/100mmHg) was calculated from blood flow (ml/min) and blood pressure (mmHg). Vasodilator kinetics were assessed by calculating the slope of the FVC response for every duty cycle between baseline and SS exercise as well as the number of duty cycles (1s contraction/2s relaxation) to reach SS. NOS inhibition reduced the kinetics of vasodilation during dynamic FA exercise (8.5 ± 0.6 vs. 11.1 ± 0.8 FVC/duty cycle; P < 0.001 vs. control) and increased the time to reach SS (25.1 ± 1 vs. 32 ± 1 duty cycles; P < 0.001 vs. control). The kinetics of vasodilation were reduced further with combined inhibition of NOS and adenosine (6.2 ± 0.8 vs. 7.5 ± 0.6 FVC/duty cycle; P < 0.05 vs. L‐NMMA alone), but not with combined inhibition of NOS and prostaglandins. Our data indicate that NOS inhibition blunts the kinetics of vasodilation during dynamic FA exercise and prolongs the time to reach SS.
Nifedipine does not open intrapulmonary arteriovenous anastomoses in healthy human subjects during exercise breathing 100% O2
Authors: Elliott Jonathan , Laurie Steven , Lovering Andrew
Published: June 2021

Intrapulmonary arteriovenous anastomoses (IPAVA) open in healthy human subjects during exercise breathing room air and can be detected using transthoracic saline contrast echocardiography (TTCE). However, during exercise in subjects breathing 100% O2, IPAVA remain closed. These data suggest that IPAVA are actively regulated, presumably via pulmonary vascular smooth muscle. One possible mechanism for the hyperoxic closure of these vessels is that the pulmonary vascular smooth muscle surrounding IPAVA could be directly regulated by the elevated PO2 of the blood. We hypothesized that blocking L‐type calcium channel activation with nifedipine would inhibit pulmonary vascular smooth muscle from contracting which would prevent the closure of IPAVA during exercise in subjects breathing 100% O2. Subjects performed cycle ergometer exercise at 50, 100, 150, and 200 W while breathing 100% O2 both before, and 30 min after taking nifedipine (20 mg, P.O.). During both bouts of exercise, IPAVA patency was determined using TTCE. Bubble scores (0–5) remained unchanged between conditions and ranged from 0–1 during all workloads. These data suggest that the effect of hyperoxia on IPAVA may be operating independently of L‐type calcium channel activation. Mechanisms of hyperoxia‐induced vasoconstriction remain elusive. Support: Giles F. Filley, Memorial Award for Excellence in Respiratory Medicine and Physiology
Reduced detrusor contractility in cavin‐1 deficient mice
Authors: Sadegh Mardjaneh Karbalaei , Uvelius Bengt , Ekman Mari , Swärd Karl , Rippe Catarina
Published: June 2021

Objective

Caveolae are omega‐shaped membrane microdomains which are abundantly expressed in the detrusor and has been shown to be of great importance for bladder function. Biogenesis of caveolae is dependent on caveolin‐1, membrane cholesterol and the recently discovered cavin‐1 protein. The aim of this study was to investigate detrusor contractility and function in cavin‐1 knockout (KO) mice.

Methods

Electron microscopy was used to visualize caveolae. Detrusor nerves were activated using electrical field stimulation (EFS).

Results and conclusion

Examination of ultrastructure confirmed the absence of caveolae in the KO mice. In response to high potassium as well as to EFS the cavin‐1 deficient mice displayed decreased force of contraction. In addition, EFS (in the presence of a muscarinic antagonist) revealed a reduced cholinergic component in the KO detrusor. Bladder to body weight was significantly increased in the KO mice suggesting bladder hypertrophy. We suggest that cavin‐1 deficiency results in impaired detrusor contractility and reduced cholinergic signalling that in turn might induce bladder hypertrophy. Supported by Swedish Research Council and the Crafoord foundation.
Differences in alcohol versus aqueous extracts of North American Ginseng in protecting against eccentric exercise‐induced muscle damage in rats
Authors: Estaki Mehrbod , Noble Earl G
Published: June 2021

The extraction process employed with North American Ginseng extracts (NAGE) is known to influence their composition and pharmacological action. Hence, the effects of different NAGE on muscle protection against acute exercise were examined. Male Wistar rats were given daily supplementation (300 mg/kg) of an Alcohol (Al) or Aqueous (Aq) ginseng extract or water placebo (P). After 14 days, each group was randomly divided into non‐exercise Control (C) or Exercise (Ex) conditions. The Ex groups ran (−14 % grade) for 1hr at a speed of 24 m/min and were sacrificed 24hr later. Plasma creatine kinase (CK) levels, structural damage (haematoxylin and eosin) and inflammation (immunostaining of the neutrophil marker, HIS48) in the soleus were analyzed. Both Al and Aq treatments significantly reduced (~18% and 25%, respectively), typical exercise‐induced rise in blood CK levels (p<0.002). However, only the Aq extract reduced morphological signs of damage (H&E) and inflammation (HIS48) (p<0.001). Steroidal saponins known as ginsenosides, found in both extracts, can reduce CK leakage by enhancing sarcolemma integrity thus lowering membrane disruption. Meanwhile, immunosuppressant actions of polysaccharides native to the Aq extract, known for their ability to attenuate over‐activation of leukocytes may explain the lack of similar protection from the Al extract. Supported by ORF grant #RE02‐049 & CIHR grant #CCT‐83029
Mathematical Model of Mixed Venous SO2 Transients at Onset of Exercise in Discrete Capillary Networks
Authors: Fraser Graham Mathew , Goldman Dan , Kowalchuk John , Ellis Christopher G
Published: June 2021

Increases in muscle O2 consumption (VO2) result in higher blood flow to accommodate changing metabolic demand. The time required for diffusion of O2 within a muscle volume to support increased VO2 causes tissue PO2 and mixed venous SO2 to lag behind VO2. The purpose of this study was to determine how diffusive transport affects time transients in venous SO2 following increases in blood flow and VO2. A finite difference model was used to simulate O2 transport in a discrete 3D microvascular network mapped from rat skeletal muscle using intravital video microscopy. Measurements were made in vivo to determine baseline simulation parameters for red blood cell supply rate (RBC SR), capillary inlet SO2, and VO2. Using the baseline solution as a starting point, exercise was simulated using simultaneous 6X step increases in VO2 and RBC SR. Tissue PO2 and capillary venous outflow SO2 (cvSO2) were recorded at 0.2s intervals until steady‐state (SS) was reached. SS mean tissue PO2 decreased from 37.2 ± 2.7 at baseline to 18.2 ± 5.9 mmHg in simulated exercise. Figure I shows the cvSO2 profile of blood as it exits the volume and the relative time course of the step change. This model demonstrates that despite instantaneous step increases in muscle VO2 and microvascular blood flow, diffusive transport of O2 in skeletal muscle imposes an observable time transient to cvSO2 following the onset of exercise.

Supported by CIHR MOP 102504 & NIH HL089125
Heterogeneity in Total Body Fat Changes After Aerobic Exercise Training is Similar in Women With Lower and Higher Amounts of Body Fat
Authors: Sawyer Brandon , Bhammar Dharini , Angadi Siddhartha , Ryan Dana , Ryder Justin , Gaesser Glenn
Published: June 2021

Aerobic exercise training in women typically results in minimal fat loss, with a significant percentage of women actually gaining body fat. We hypothesized that heterogeneity in body fat changes after training would be independent of initial % body fat. Seventy‐three non‐vigorously active women (age: 30.5 ± 7.9 yrs; ht: 164.7 ± 7.5 cm; wt: 67.2 ± 14.4 kg; %fat: 37.5 ± 8.7) underwent Dual X‐ray Absorptiometry (DXA) before and after a 12‐wk exercise training program of treadmill walking 3 days/wk for 30 min at 70% of VO2max. For all women combined, % body fat was reduced modestly after training (−0.53%; P = 0.02). Women with the lowest baseline body fat (LF: n=37; %fat = 30.2 ± 5.2%) and highest baseline body fat (HF: n=36; %fat = 44.5 ± 4.8%) did not differ with regard to changes in %fat (LF = −0.28 ± 1.6%; HF = − 0.78 ± 2.1%; P = 0.27) or total fat mass (LF = −0.04 ± 1.3 kg; HF = −0.76 ± 2.5 kg; P = 0.14). Leg fat was reduced by more in HF (HF = −0.93 ± 2.9 kg; LF = +0.21 ± 1.8 kg; P < 0.05), indicating greater reduction in gynoid fat pattern in HF (HF = −1.88 ± 2.0%; LF = − 0.04 ± 2.0%; P < 0.01). Both groups had marked heterogeneity in fat changes, with 50% of LF and 39% of HF gaining total body fat after training. Our results suggest that regardless of initial body fat, aerobic exercise training in women is characterized by “losers” and “gainers” in total body fat. Women with high initial body fat may be more likely to lose lower body fat and have a reduced gynoid fat pattern.
Control of muscle VO2 kinetics: a role for metabolic rate?
Authors: Wüst Rob CI , McDonald James R , Ferguson Brian S , Sun Yi , Rogatzki Matthew J , Spires Jessica , Kowalchuk John M , Gladden L Bruce , Rossiter Harry B
Published: June 2021

Control of skeletal muscle oxygen uptake (VO2) kinetics is mediated by ADP feedback, oxygen and substrate delivery and mitochondrial content. Controversy remains whether mitochondrial enzyme activation at exercise onset contributes to this control. We tested the hypothesis that VO2 kinetics would be speeded by increased metabolic rate, in pump‐perfused, tetanically‐stimulated muscle (i.e. independent of O2 delivery and muscle recruitment dynamics). The gastrocnemius of 6 anaesthetized, ventilated dogs was isolated and the tendon attached to a force transducer. Blood flow was pump‐controlled at a constant flow (1.09 ± 0.25 L.kg−1.min−1; mean ± SD). Isometric tetanic contractions (50 Hz; 200 ms duration) were elicited via supramaximal sciatic nerve stimulation. Metabolic rate was manipulated using 3 min of stimulation at 0.33 Hz (S1) immediately followed by 3 min at 0.67 Hz (S2); also following prior contractions at 0.67 Hz and 2 min recovery (PS1, PS2). Muscle VO2 was determined contraction‐by‐contraction. τVO2 was lower in S1 (11.4±2.4 s; p<0.05) than S2 (17.6±2.9 s), PS1 (16.6±4.6 s) and PS2 (16.1±2.5 s). Unexpectedly VO2 kinetics were fastest at the lowest stimulation frequency without prior contractions. This implies that any putative role of mitochondrial activation in VO2 kinetic control may be countered by a reduction in cellular energy state (e.g. less negative ΔGATP). Supported by BBSRC: BB/F019521/1
Influence of daily ethanol injection on compensatory hypertrophy in the rat
Authors: Blank Jason Michael , Fugle Hannah M , Malcolm Colin J , Mendoza Doris , Schaller Emma K , Van Houten Erik J
Published: June 2021

Acute ethanol exposure inhibits muscle protein synthesis, while chronic ethanol exposure causes muscle wasting in humans and laboratory animals. In contrast, resistance exercise increases muscle protein synthesis and thus muscle mass, but few studies have addressed the interaction of ethanol and exercise. To assess this interaction, we removed the right gastrocnemius from Sprague‐Dawley rats (n=23) and then injected the animals with ethanol (75 mmol/kg) or an equivalent volume of saline daily for 3 weeks. Surgically overloaded plantaris muscles were 85% larger than muscles from contralateral control legs in saline‐treated animals and 94% larger than contralateral controls in ethanol‐treated animals. The relative mass of the non‐surgical plantaris was not significantly different in saline‐ and ethanol‐treated animals (0.85 ± 0.02 g/kg for ethanol vs. 0.89 ± 0.03 g/kg for saline, p=0.10). Ethanol also had no effect on relative mass of the overloaded plantaris (1.67 ± 0.09 g/kg for ethanol vs. 1.65 ± 0.06 g/kg for saline, p=0.81). These results indicate that a single daily dose of ethanol is unable to prevent overload‐induced hypertrophy in skeletal muscle. This work was sponsored by the Department of the Navy, Office of Naval Research, under Award # N00014‐11‐1‐0359.
Diaphragm Contractile Function Differs Among Murine Strains
Authors: Stasko Shawn , Reid Michael B
Published: June 2021

Murine diaphragm fiber bundles are routinely used to study physiologic properties of skeletal muscle. Based on pilot data, we hypothesized that contractile function differs among strains in healthy mice.

Methods

We measured specific force (N/cm2) and fatigue characteristics in diaphragm fiber bundles from age‐matched male ICR, C57BL/6, and DBA‐1 mice. Fiber bundles were studied in vitro (optimal length, 37°C, n=4/group) using a standard force‐frequency protocol (1, 15, 30, 50, 80, 120, 180, 250, 300 Hz). Fatigue was induced using repeated submaximal tetanic contractions (40 Hz, 500 ms trains; 0.5 trains/s) for 5 min.

Results

Among groups, ICR muscle generated higher maximal forces (26.4 N/cm2 ± 0.3 SE) than C57BL/6 (21.1 ± 0.3) or DBA (14.7 ± 0.2). Relative force (as % maximal) did not differ among groups at any stimulus frequency. During the fatigue protocol, force fell less in DBA muscle (5.9 to 3.0 N/cm2; −50% decrement) than ICR (8.8 to 2.7; −69%) or C57BL/6 (8.3 to 3.0; −69%).

Conclusion

Specific force and fatigue characteristics differ among diaphragm fiber bundles of ICR, C57BL/6, and DBA‐1 mice.

These data suggest that diaphragm contractile properties differ among murine strains. Supported by NIH grant HL08341 (T32 training fellowship).
Mouthpiece Use Reduces Post Exercise Serum Cortisol Levels
Authors: Dudgeon Wes D. , Scheett Timothy P. , Buchanan Larry A. , Strickland Ashley E. , Garner Dena P.
Published: June 2021

Recent work has shown that a lower jaw mouthpiece (MP) can decrease lactate, improve airway dynamics and decrease salivary cortisol following exercise. This study aimed to determine if wearing a lower jaw MP (ArmourBite Technology) altered serum cortisol in 13 collegiate‐aged males during or after physical work. At least 7 days following one repetition maximum (1RM) testing, subjects completed 2 trials, separated by 7 days, of 6 sets x 10 repetitions of back squats with 2 min rest between sets. Load was set at 80% of each subject's 1RM and resistance was adjusted during testing so subjects completed 10 repetitions during each set. Treatment order was randomized and blood was sampled: pre exercise, after 3 sets (mid), post exercise, 30, 60 and 120 min post exercise. Each sample was labeled and frozen for determination of cortisol (ìg/dL) via ELISA (ALPCO Diagnostics, Salem, NH). There was no difference in pre exercise cortisol between groups (MP: 21.55±9.82 vs 25.58±10.88). The MP group had lower (P<.05) cortisol levels at mid (MP: 19.39±6.90 vs 27.84±14.55) and post 30 (MP: 22.91±8.47 vs 31.81±10.79), while no differences existed immediately post exercise, post 60, or post 120. These results confirm in serum that the use of lower jaw MPs attenuates the cortisol response following strenuous physical exercise. This suggests that the use of a MP may aid in post exercise recovery and may diminish the stress of physical exercise.
Cellular mechanisms of tendon‐muscle crosstalk
Authors: Abreu Eduardo , Romero-Suarez Sandra , Brotto Leticia , Mo Cheng Lin , Brotto Marco
Published: June 2021

Although transmission of mechanical load is the most obvious relationship between muscles, tendons and bones, new evidence has shown that these tissues might also communicate through biochemical factors. Previous results from our lab suggest the existence of crosstalk from bone to muscle cells and vice‐versa. To investigate a possible crosstalk between tendons and muscles, we tested the hypothesis that conditioned media from tenocytes (CMT) would exert noticeable effects on myogenic differentiation. C2C12 myoblasts were treated with differentiation (DIFF = DMEM + 2.5% horse serum, control media) or DIFF + 10% CM‐T media for up to 7 days. CM‐T was obtained from 90% confluent cultures of tenocytes retrieved from ex‐vivo explants of C57Bl6 WT mouse Flexor Digitorum Brevis (FDB) tendon. CM‐T had a profound effect to promote myogenic differentiation. There were significant increases in mean myotube area and fusion index, but not in number of myotubes observed at 100X magnification. Furthermore, CM‐T caused the upregulation of myogenic regulatory factors (MRF), including MyoD1, and of genes related to the Calcineuring‐NFAT pathway. These findings are the first evidence of a potential biochemical crosstalk between tendons and muscles after development. Support: NIH RC2 AR058962 and Missouri Life Sciences Research Board to MB.
Nitric oxide activates hypoglossal motoneurons by cGMP‐dependent inhibition of TASK channels and cGMP‐independent activation of HCN channels
Authors: Wenker Ian Christopher , Chen Xinnian , Liu Hattie , Horner Richard , Mulkey Daniel Kent
Published: June 2021

Nitric oxide (NO) is an important signaling molecule; however mechanism(s) underlying NO modulation of motoneurons (MNs) remain obscure. Here, we used a combination of in vivo and in vitro recording techniques to examine NO modulation of MNs in the hypoglossal motor nucleus (HMN). Microperfusion of DEA (NO donor) into the HMN of anesthetized adult rats increased genioglossus muscle activity. In the brain slice, whole‐cell current‐clamp recordings from hypoglossal MNs show that exposure to DEA depolarized membrane potential and increased responsiveness to depolarizing current injections. Under voltage‐clamp conditions, we found that NO inhibited a Ba2+‐sensitive background K+ conductance and activated a Cs+‐sensitive hyperpolarization‐activated inward current (Ih). The NO‐sensitive K+ conductance exhibited properties similar to TWIK‐related acid‐sensitive K+ (TASK) channels and was blocked by ODQ (soluble guanylyl cyclase antagonist) and KT5823 (PKG antagonist). In Ba2+, NO activated both the instantaneous (Iinst) and time‐dependent (Iss) components of Ih. Interestingly, at more hyperpolarized potentials NO preferentially increased Iinst, and this was retained in ODQ but blocked by a cysteine‐specific oxidant. These results suggest that NO activates hypoglossal MNs by cGMP‐dependent inhibition of a TASK‐like current and S‐nitrosylation‐ dependent activation of Ih.
Neonatal gender contributes to respiratory pattern variability re‐establishing stable rhythmogenesis following hypoxia
Authors: Garcia Alfredo J , Malashchenko Tatiana , Rotem-Kohavi Naama , Ramirez Jan-Marino
Published: June 2021

Gender affects many aspects of neuronal control and has been implicated in the etiology of SIDS because males are more likely to die of SIDS than females. This study tests the hypothesis that gender influences neonatal respiratory patterns following hypoxia. Experiments in CD1 mice (P10–13days) reveal that recovery from peak post‐hypoxia depression of respiratory rate is significantly delayed in males. Recordings from brainstem slices containing the preBötzinger complex (preBötC) show that male rhythmogenesis recovers slower from hypoxia compared to female rhythmogenesis. Recovery time in males correlated to baseline respiratory frequency before hypoxia, which was not the case in females. We employed computational modeling to investigate the underlying mechanisms. Based on multi‐array and intracellular recordings from spontaneously active respiratory networks, we modeled the preBötC as a sparsely connected network containing different types of respiratory neurons including, various pacemaker, tonically active and silent neurons. Our modeling indicates that different neuronal types have different recovery profiles, and that recovery time and frequency dependency are dependent on network composition. This approach allows us to make specific predictions which neurons or neuronal properties could explain observed gender differences. Our data indicate that even when intrinsic membrane properties of individual neurons of the population are conserved the observed dimorphic differences could be explained by differences in network composition. Ongoing experiments aim at testing these hypotheses in the respiratory network.
Genome‐Wide P‐element Screen for Hypoxia Tolerance Genes in Drosophila melanogaster
Authors: Haddad Gabriel G. , Azad Priti
Published: June 2021

Hypoxia occurs during normal conditions (high altitude) or during pathologic states (sickle cell anemia). Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. To identify the genes involved in hypoxia tolerance, we screened P‐element insertion lines generated by Gene Disruption Project. For hypoxia tolerance, we screened for a) eclosion rates after following the development of embryos placed in 5% O2 to eclosion and adulthood, b) number of adult flies surviving after eclosion. Out of 2367 lines screened, 44 P‐element lines had eclosion rates significantly higher (>70% eclosion) than the controls (eclosion rate ~7–8%) under hypoxia. The molecular function of these lines ranged from cell cycle regulation, DNA or protein binding, ATPase activity, and transcriptional co‐regulators. In this screen, we found certain interesting candidate genes such as sec8, cyclin E, osa, l(3)mbn, lqf which show tremendous hypoxia tolerance during all stages of the developmental cycle. Furthermore, we employed the UAS–Gal4 system to further dissect the role of these genes ubiquitously or in specific tissues in vivo. Interestingly, we found that regulation by single genes, such as osa can play an important role in survival and development during hypoxia.

This work has been supported by NIH grant (RO1 NS037756) to Dr. Gabriel Haddad.
Screening of siRNA to identify the genes associated with vascular collapse when exposed to Yersinia pestis
Authors: Hoke Allison Violet , Gautam Aarti , Filippov Andrey , Amemiya Kei , Hammamieh Rasha , Jett Marti
Published: June 2021

Yersinia pestis, the causative agent of plague, upon leukocyte activation leads to the systemic, often fatal, vascular collapse and end‐organ failure. To investigate genes associated with vascular collapse, we studied the permeability of human lung microvascular endothelial cells (HMVEC‐L) co‐cultured with peripheral blood mononuclear cells (PBMCs) in the presence of bacteria. The monolayer's permeability was measured using fluorescein dextran in a 96‐transwell format. To identify host factors, a primary screen of ~400 siRNAs against drugable target genes was performed and the cells were plated for monolayer formation. The co‐culture of HMVEC‐Ls with Y. pestis (CO92pgm−) infected PBMCs had no effect on permeability. About 10 genes that lead to increased permeability when knocked down were identified in the initial screen and need to be confirmed. In another approach to identify siRNA's that block vascular leakage, a pathogenic strain of bacteria that leads to vascular collapse was used. Experiments have been performed to optimize the Y. pestis infected PBMC/HMVEC‐L ratio to determine the best conditions that lead to an increase in vascular permeability. This information and the siRNA knock down results will help identify genes with a potential for therapeutic roles. Research support is through DTRA, TMTI.DRUG.02.10.WR.023.
Coordinated microRNA/gene networks central to nutrient responsiveness in the 0.9G baboon fetal kidney
Authors: Cox Laura A , Glenn Jeremy P , Lange Kenneth P , Nijland Mark J
Published: June 2021

MicroRNAs are environmentally sensitive gene regulators that play roles in proliferation and differentiation. We hypothesized that miRNAs underlie fetal baboon kidney transcriptome response to nutrient restriction (NR) via coordinated miRNA/gene networks. We used in silico methods to identify NR sensitive miRNAs in fetal kidneys.

Differentially expressed genes from 0.9G baboon kidney [CTR, n=3;NR, n=3,70%CTR] were assessed for miRNAs with TargetScan based ID by: genomic location congruency; enrichment analysis; and network analysis to ID hub genes and networks predicted to be regulated by miRNAs.

Genomic location congruency identified 145 miRNA families; enrichment analysis ranked 55 families; network analysis revealed 7 families with target sites in 3 or more hub genes. The mir‐27 family has target sites in 7 hub genes and 35 network genes, many transcription factors and all down regulated in NR.

Only network analysis provided a directed approach to selecting miRNAs that may coordinately regulate gene expression. Results suggest that miRNAs regulate NR responsiveness in renal development via transcription factors and reveal potential transcriptome/epigenome interactions fundamental to renal development.
Bicarbonate‐dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu‐3
Authors: Shan Jiajie , Liao Jie , Huang Junwei , Robert Renaud , Palmer Melissa L. , Fahrenkrug Scott C. , O'Grady Scott M. , Hanrahan John W.
Published: June 2021

Anion transport by the airway is defective in cystic fibrosis (CF), but its mechanisms and roles in fluid secretion are not well understood. We studied Cl− and HCO3− fluxes across Calu‐3 cells, calculated the equivalent short‐circuit current (Ieq) under open circuit conditions, and measured fluid secretion by monolayers.

Forskolin stimulated Ieq, JHCO3− and fluid secretion. Bumetanide reduced secretion by ~20%, but had no effect on HCO3− transport. Although secretion was abolished in Na+‐free or HCO3−‐free solution, significant JHCO3− was maintained in Cl−‐free solution. DIDS was inhibitory in normal and Cl−‐free solutions. Using apical permeabilization we demonstrated an exchange of intracellular HCO3− for basolateral Cl− or NO3−, but not for gluconate. Adding basolateral NO3− to low‐Cl− medium restored fluid secretion. Acetazolamide greatly inhibited anion transport.

Our data show that basolateral anion exchange plays an important role in Cl− loading and fluid secretion. NBC mediates HCO3− entry and carbonic anhydrase contributes significantly to the intracellular accumulation of HCO3−. Although HCO3− is essential for fluid secretion, most HCO3− is recycled to the basolateral side in exchange for Cl−, thus fluid transport depends mainly on JCl− rather than JHCO3−. HCO3− secretion persists in Cl−‐free solution and is sensitive to CFTR blockers, suggesting apical HCO3− exit is mediated by CFTR.
Sorting protein‐related receptor SorLA is involved in regulation of renal Na+‐K+‐2Cl‐cotransporter through interaction with an isoform of calcineurin phosphatase
Authors: Borschewski Aljona , Willnow Thomas , Dathe Christin , Paliege Alexander , Ferreri Nicholas , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Activity of the Na+‐K+‐2Cl‐cotransporter (NKCC2) of the thick ascending limb (TAL) is dependent on its N‐terminal phosphorylation. Sorting protein‐related receptor SorLA was shown to facilitate this process. To clarify the interference between SorLA and NKCC2 we have evaluated the profile of relevant kinases and phosphatases in TAL of SorLA‐deficient (Sorla−/−) compared to wild‐type (WT) mice.

SorLA‐deficiency was associated with nearly absent NKCC2 phosphorylation (−97%). Levels of the SPAK‐OSR1 kinases were not changed while the abundance of the calcineurin phosphatase PP2BAβ was significantly increased in SorLA‐deficiencient animals (+201%). Confocal analysis revealed apical accumulation of the phosphatase in TAL of Sorla−/− mice. The interaction of NKCC2 with PP2BAβ was verified by co‐immunoprecipitation. Application of a calcineurin inhibitor cyclosporine in cultured TAL cells induced an increase of NKCC2 phosphorylation.

Our results establish PP2BAβ as a relevant phosphatase responsible for the dephosphorylation of NKCC2 and provide evidence that SorLA may mediate degradation of PP2BAβ thus facilitating the phosphorylation of the transporter.
E2F1 is a potential novel regulator of liver fibrosis by targeting Egr‐1 through nuclear receptor SHP
Authors: zhang yuxia , wang li
Published: June 2021

Objective

Egr‐1 is a critical regulator of liver fibrosis. However, the transcriptional network that governs Egr‐1 expression remains largely unknown. This study aimed at identifying novel transcriptional factors controlling Egr‐1 expression and liver fibrosis.

Methods

Luciferase reporter and ChIP assays were used to elucidate the mechanisms. Mouse models of liver fibrosis induced by bile‐duct ligation (BDL), CCl4, or 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine were used as in vivo models.

Results

The SHP‐/‐ mice showed increased fibrosis in response to BDL, which was reversed in hepatocyte SHP‐overexpressed mice. The Egr‐1 promoter was activated by E2F1, and the activation was abrogated by co‐expression of SHP and EID1 in both human hepatoma Huh7 and stellate cells LX2. ChIP assays confirmed the association of E2F1 and SHP with the Egr‐1 promoter, and GST pull‐down confirmed a direct interaction between E2F1 and SHP. Interestingly, the loss of E2F1 had no effect in CCl4‐intoxicated mice, but significantly decreased liver fibrosis of the biliary type. Importantly, E2F1 and Egr‐1 expression were dramatically increased in human cirrhotic liver, which was inversely correlated with diminished SHP expression.

Conclusions

Egr‐1 expression is regulated by a transcription network involving E2F1 and SHP. E2F1 may function as a novel fibrogenic gene. Supported by T32CA092347 (Y.Z) and DK080440 (L.W).
17β‐Estradiol relaxes cholecystokinin (CCK) and KCl‐induced tension in female guinea pig gallbladder strips by inhibiting extracellular Ca2+ entry
Authors: Kline Loren , Karpinski Edward
Published: June 2021

17β‐estradiol (E2) induced a concentration‐dependent relaxation of CCK‐ or KCl‐induced tension. When the response to E2 between strips from young guinea pigs and strips from those in late pregnancy were compared there was no significant difference in the response to 50 or 100 μM E2; but, 10μM caused a significant (p<0.05, 19.3±5.4 vs. 25.9±2.7%, n=7) increase in the amount of relaxation in strips from pregnant guinea pigs. PKA inhibitor 14–22 amide myristolated had no significant effect on the E2‐induced relaxation. Treatment with 2‐APB, an inhibitor of IP3 induced Ca2+ release, produced a significant (p<0.01) increase in the amount of E2‐induced relaxation when either CCK or KCl were used. Neither KT5823, a PKG inhibitor, nor L‐NMMA, a NO synthase inhibitor, had a significant effect on the E2‐induced relaxation. Bisindolymaleimide IV and chelerythrine Cl−, PKC blockers, were used in combination with no significant effect on the amount of CCK‐induced tension, but significantly (p<0.01) increased the amount of E2‐induced relaxation. When E2 was added to the chambers 3 min prior to either CCK or KCl, a significant (p<0.001) decrease in the amount of tension was observed (CCK, 0.78±0.08 vs. 0.36±0.03g, n=5; KCl, 1.09±0.06 vs. 0.20±0.02g, n=6). The inhibition of extracellular Ca2+ entry mediated E2‐induced relaxation of CCK‐ and KCl‐induced tension in female guinea pig gallbladder strips.
Circulatory Estradiol Influences Gastric Emptying, Nitrergic Relaxation via Tetrahydrobiopterin (BH4, a cofactor nNOS dimerization) in a female Mouse, Model of Chronic Hypoestrogenemia
Authors: Gangula Pandu R , Ravella Kalpana , Srinivasan Shanthi , ChakradhariI Sharan , Al-Hendy Ayman , Hale Ashley , Channon Keith
Published: June 2021

Gastroparesis (delayed gastric emptying) affects predominantly females more than males specifically due to loss of nitrergic (neurons release nitric oxide) dependent stomach motility function. We hypothesize that reduced levels of estradiol‐17β (E2) down‐regulates BH4 biosynthesis and therefore nNOS mediated gastric motility is impaired in a mouse model of chronic estrogen deficiency, follicle stimulating hormone receptor knock‐out female mice (FORKO). In‐bred 12 week old female FORKO mice were obtained from our local FORKO breeding colony. In FORKO, compared to wild type (WT) stomachs we find, 1) greater reduction in nitregic relaxation (WT vs. −/‐; −0.28±0.01 vs. −0.16±0.01); 2) increased nNOS dimerization (WT vs. −/‐; 0.34±0.05 vs. 0.48±0.02) (p<0.05); 3) decreased GTPCH1 (WT vs. −/‐; 2.4±0.07 vs. 1.8±0.14) and DHFR (WT vs. −/‐; 0.24±0.03 vs. 0.02±0.00) and 4) increased oxidized biopterin levels and decreased ratio of BH4 vs. BH2+B (p<0.05). Diabetes induction by HFD significantly (p<0.05) reduced both gastric GTPCH1 and nNOSα protein expression. Chronic estrogen deficiency alters both BH4 and nNOS system and thus contributes for the development of gastroparesis in female. In addition, HFD induced diabetes impaired both GTPCH1 and nNOSα protein expression. Our studies may offer a potential therapeutic approach to treat gastroparesis in both type 1 and type 11 diabetes women. Funding: grants from RCMI, DRTC, R21 (PG).
The Metabolism of Polymethoxyflavone and Its Implication in Colon Cancer Inhibition
Authors: Xiao Hang , Zheng Jinkai , Song Mingyue , Zhong Zhimei , Wang Mingqi
Published: June 2021

We have previously reported inhibitory effects of 5‐hydroxy nobiletin (5HN, a unique citrus flavonoid) on colon cancer. Herein, we investigated colon metabolism of 5HN in rodents and its implication in colon cancer inhibition. Using LC‐MS and NMR, we identified 3 major metabolites of 5HN in GI tract of mice and rats, i.e. 3′,4′,5‐trihydroxy nobiletin (M1), 3′,5‐dihydroxy nobiletin (M2), and 4′,5‐dihydroxy nobiletin (M3). In small intestine mucosa, about 55% of 5HN and 95% of its metabolites were in conjugated forms (glucuronide and sulfate). In contrast, only 10% of 5HN and 25% of metabolites were in conjugated forms in colonic mucosa. This is presumably due to de‐conjugation by colonic microflora. 5HN and its metabolites in conjugated forms lacked inhibitory effects on colon cancer cells in comparison with their free forms. In the mice fed with 0.05% 5HN in diet for 1 week, the levels of free 5HN and its metabolites ranged from 1~10 μg/g tissue in colonic mucosa (equivalent to 3–26 μM, assuming 1 gram of tissue occupies 1 mL of volume). All three metabolites were more potent in inhibiting colon cancer cell growth than 5HN, especially, M2, e.g., IC50s of M2 were 50~100‐fold lower than those of 5HN. Our results provide a strong basis for using 5HN to inhibit colon cancer due to its desirable colonic metabolism profile.
Mouse betaine homocysteine S‐methyltransferase deficiency reduces body fat
Authors: Teng Yawen , Ellis Jessica M , Coleman Rosalind A , Zeisel Steven H
Published: June 2021

Betaine homocysteine S‐methyltransferase (BHMT) catalyzes the conversion of homocysteine to methionine and is predominantly found in the liver. Mice with the gene encoding Bhmt deleted (Bhmt−/−) had reduced adiposity, smaller adipocytes, better glucose tolerance and enhanced insulin sensitivity than the controls. The reduced adiposity in Bhmt−/− mice was not due to alterations in food intake, fat absorption, or thermogenesis. Compared to the controls, Bhmt−/− mice had increased energy expenditure, reduced mobilization of lipid from the liver to adipose tissue, decreased lipid synthesis within adipocytes, and enhanced whole‐body glucose oxidation. In addition, Bhmt−/− mice had altered concentrations of betaine, choline, bile acids, thyroid hormones, and fibroblast growth factor 21, all of which have been associated with reduced adiposity. We are currently studying which of these factors is the underlying mechanism of the reduced adiposity observed in Bhmt−/− mice. Research was supported by grants from the NIH DK55865.