bluuebunny/crossref_metadata_2025 huggingface dataset:
Markers of Mitochondrial Biogenesis following 3 days of Cycling
Authors: Dumke Charles L , Nieman David C , Davis J Mark , Murphy E. Angela , Carmichael Martin D , Henson Dru A , Quindry John C , Utter Alan C , Triplett N. Travis , McAnulty Steven R , McAnulty Lisa S
Published: June 2021

Exercise increases mRNA for several genes involved in mitochondrial biogenesis. Less is known about how these genes respond to several successive days of intensive exercise.

PURPOSE:

To examine the effects of 3 h of intensive cycling over 3 successive days on gene expression in humans.

METHODS:

Twenty trained cyclists were tested for VO2max (54.7±1.1 ml.kg‐1.min‐1). The subjects cycled at 57% wattsmax for 3 h using their own bicycles on CompuTrainer™ Pro Model trainers (RacerMate, Seattle, WA) on 3 successive days. Muscle biopsies were obtained from the vastus lateralis pre‐ and post‐exercise on days 1 and 3. Muscle samples were analyzed (2(time) X 2(day) repeated measures ANOVA) for gene expression of cytochrome C, peroxisome proliferator receptor gamma coactivator‐1 alpha (PGC‐1A), citrate synthase, and Sirt‐1.

RESULTS:

Of the genes analyzed the following showed significant increases pre‐post 3 h rides: cytochrome C (P=0.03), PGC‐1A (P<0.001), and Sirt‐1 (P=0.05). The following genes showed significant effects from days 1 to 3: cytochrome C (P<0.001) and citrate synthase (P=0.009).

CONCLUSION:

Three successive days of intensive cycling resulted in upregulation of mRNA for mitochondrial enzymes and biogenesis markers in these trained subjects. These data contribute to the understanding of mitochondrial enzyme expression during prolonged exercise training.
Increased duration of involuntary exercise has a greater effect on GDNF protein expression in skeletal muscle
Authors: Czarnopys Monica Janine , Kinnell Kyle R , Smith Jennifer S. , Spitsbergen John M.
Published: June 2021

Glial cell line‐derived neurotrophic factor (GDNF) is a neurotrophic factor found in skeletal muscle that affects peripheral motor neurons. The purpose of our experiment was to examine changes in GDNF protein expression in skeletal muscle from rats that have undergone different durations of exercise. GDNF protein content of skeletal muscle was examined following involuntary exercise on a running wheel for 2, 4, and 6 weeks. Muscles examined included diaphragm (dia), pectoralis (pec) and extensor hallucis longus (ehl). GDNF protein content was measured via enzyme‐linked immunosorbant assay. The results show that pec and ehl muscles responded to exercise with an early decrease in GDNF content (2 weeks), followed by a return to control levels after 4 and 6 weeks of exercise. Dia muscle showed a significant increase in GDNF protein content following 6 weeks of training. If GDNF expression in skeletal muscle is regulated by physical activity this could help to explain beneficial effects of exercise on motor nervous system structure and function. This work was supported by NIH grant 1 R15 AG022908‐01A2, The Monroe‐Brown Foundation, Western Michigan University, and MSU‐KCMS.
Effects of post exercise supplementation with protein and carbohydrate on lean tissue mass gains during 70 days of resistance training in healthy males and its association with mediators of the molecular regulation of muscle mass
Authors: Greenhaff Paul Leonard , Karagounis Leonidas G. , Pittas George , Layfield Robert
Published: June 2021

We studied the effect of 70 days of resistance training (RT), or RT plus protein and carbohydrate ingestion, on lean tissue and fat mass gains and mediators of anabolic signalling and MAFbx protein expression in human muscle. Untrained subjects performed supervised RT (3 times/week) for 70 days at 80% of 1 rep. max. (reevaluated every 2 weeks and adjusted accordingly). Immediately after each training session, subjects consumed 3g of flavourings (Control, n=8) or 50g whey protein and 50g dextrose (ProDex, n=8) in a non‐isocaloric solution. Body composition (DEXA) and a quadriceps biopsy sample were obtained at baseline and day (d)2, d35 and d70 (all 24h following training). Lean tissue mass increased from baseline by d35 and d70 in Control (P<0.01, P<0.001, respectively) and ProDex (P<0.001, P<0.001, respectively), but there was no difference between groups. Similarly, fat mass declined in both groups with training, with no difference between groups. Compared to Control, ProDex was associated with increased phosphorylation of Akt/PKB (d2, P<0.01; d70, (P<0.05), mTOR (d70, P<0.05), p70S6k (d70, P<0.001) and decreased expression of MAFbx (d2, P<0.01) protein. Post‐exercise ingestion of ProDex over 70 days RT had no additional effect on body composition than training alone, despite differences in mediators of the molecular regulation of muscle mass.

This study was funded by Iovate Inc.
Twenty weeks of whole body resistance training improves the response of leg blood flow to feeding at rest and after exercise
Authors: Phillips Bethan , Wilkes Emilie , Smith Kenneth , Baker Margaret , Rennie Michael
Published: June 2021

There is little information on how resistance training affects muscle blood flow responses at rest to a meal and to prior exercise. Therefore, we studied the response to feeding with or without acute resistance exercise (RE) before and after 20 w of whole body RE training (thrice weekly, 70% 1RM, with 1RM re‐assessments monthly). Before and after training, 10 subjects, 8 male and 2 female (21–73 y), (BMI >18<28) performed 6×8 isotonic unilateral leg extensions at 75% 1RM before intermittent feeding (at 4.25×BMR for 2.5 h). Femoral artery blood flow was measured by Doppler ultrasound at rest in both legs throughout.

Before training, feeding alone or exercise with feeding increased resting blood flow. After training, blood flows in all conditions were increased. Whole body resistance training increases the responses of resting leg blood flow to feeding alone or feeding after exercise. Such an adaptation is likely to increase nutrient delivery to leg muscle and help maintain muscle mass, as well as potentially increasing insulin sensitivity.

Supported by UK BBSRC ((BB/XX510697/1 and BB/C516779/1) and a European Union EXEGENESIS program grant.
Effects of Interrupted Resistance Training on Bone Mineral Density in Growing Rats
Authors: Godfrey James K. , Kayser Brandon D. , Gomez Gregory V. , Bennett Jason , Jaque S. Victoria , Sumida Ken D.
Published: June 2021

The purpose of this study was to examine the efficacy of interrupting a daily resistance training program for enhancing bone mineral density (BMD) in growing male rats. We compared a resistance training program where the exercise was uninterrupted (i.e., continuous repetitions) against a resistance training program where the exercise was interrupted (2X during a training day). The total volume of work performed between the two resistance trained groups was equivalent. 23 male rats were randomly divided into Control (Con, n = 8), uninterrupted resistance training (UT, n = 8), or interrupted resistance training groups (IT, n = 7). The UT and IT groups were conditioned to climb a vertical ladder with weights appended to their tail 3 days/wk for 6 wks. All exercised animals initially carried 30% of their body weight (BW) and progressed to 150% BW. Serum osteocalcin (OC), urinary deoxypyridinoline (DPD), and left tibial BMD were determined in all groups. OC (ng/ml) was significantly greater for both UT (45.7 ± 1.0) and IT (46.3 ± 1.7) compared to Con (37.9 ± 1.4). DPD was not significantly different between groups. Left tibial BMD (g/cm2) was significantly greater for both UT (0.237 ± 0.003) and IT (0.238 ± 0.002) compared to Con (0.223 ± 0.001). The results indicate that both resistance training programs were equally effective in eliciting an osteogenic response that culminates in a significant elevation in BMD in growing animals.
Does Post Exercise Protein Reduce Total Protein Needs for Optimal Skeletal Muscle Responses to Resistance Training in Older Adults?
Authors: Lee Teak V , Lee Chang Woock , Gasier Heath G , Chikani Gentle , Chen Vincent , Riechman Steven E
Published: June 2021

Purpose: Age‐related muscle and strength loss, Sarcopenia, is a significant problem in the elderly population but may be restored through adequate protein intake and resistance exercise training (RET). The amount and timing of protein consumption may potentially play an important role in skeletal muscle responses to RET. In this study, we tested the hypothesis that post‐exercise protein was sufficient to elicit the beneficial effects, with higher protein intakes unnecessary. Methods: 30 older, generally healthy men and women (age 50–69) performed 12 wks of full‐body RET. Following the sessions, subjects received a whey/egg protein supplement (0.2 g/kg lean mass). In addition, subjects were asked to complete 48 food records over 12 wks. Body composition (DEXA), 1‐RM and power tests were conducted before and after the training. Results: Mean dietary intake of protein during the 12 wks (g/kg) was significantly (P<0.01) correlated to percent change in lean mass (r=0.394). Percent change in lean mass for low (0.7 g/kg), moderate (0.9 g/kg) and high (1.1 g/kg) protein intake per day was 2.3±0.8%, 3.6±0.7%, and 5.1±0.8%, respectively. Conclusion: Contrary to our hypothesis and previous studies, variability in total daily protein intake did influence muscle gain with RET when post‐exercise protein was administered.

This study was supported by the US Poultry and Egg Association.
Ethinyl estradiol suppress tendon collagen synthesis in response to exercise
Authors: Hansen Mette , Koskinen Satu , Petersen Susanne Germann , Dossing Simon , Frystyk Jan , Flyvbjerg Allan , Kjaer Michael , Langberg Henning
Published: June 2021

We aimed to investigate whether oral contraceptives (OC), resulting in enhanced circulating level of ethinyl‐estradiol, influences tendon collagen metabolism at rest and after exercise. Healthy young women, OC‐users (n=11) and control (n=12), were studied 24h following one‐legged kicking exercise (EX) for 60 min at 67 % of Wattmax. Microdialysis catheters were placed anterior to the patellar tendon of both legs, and interstitial fluid was collected and analyzed for the amino‐terminal propeptide of type I collagen (PINP), a marker of tendon collagen synthesis. An exercise‐induced increase in tendon synthesis was observed in control (PINP: Rest: 25.3 ± 11 (mean ± SD) ng ml−1 vs. EX: 48.3 ± 28 ng ml−1, P < 0.05), but not in OC‐users (EX: 27.4 ± 16 ng ml−1 vs. Rest: 31.5 ± 20 ng ml−1). Insulin‐like growth factor I (IGF‐I) both in serum and in the interstitial tendon tissue fluid was significantly lower in OC‐users compared to control, whereas IGFBPs were significantly higher, which in combination with lower IGF‐I. In conclusion, OC seems to have an inhibiting effect on tendon synthesis in response to exercise either directly or indirectly by a reduction of the bioavailability of IGF‐I.
Influence of resistance exercise on human patellar tendon structural and regulatory gene expression
Authors: Sullivan Bridget E , Carroll Chad C , Jemiolo Bozena , Trappe Scott W , Trappe Todd A
Published: June 2021

The tendon is mainly composed of collagen surrounded by a matrix of proteoglycans that are regulated by enzymes called matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Studies have shown that acute exercise stimulates collagen turnover, but little is known about the influence exercise has on other components of the tendon. Therefore, we measured the gene expression (mRNA) levels of the tendon's main proteins (collagen type I and collagen type III), proteoglycans (decorin, biglycan, fibromodulin, and versican), and regulatory enzymes (MMP‐2, MMP‐9, MMP‐3, and TIMP‐1) at rest and following resistance exercise (RE). Patellar tendon biopsy samples were taken from six individuals (3M, 3F) before and 4h following a bout of RE (3 sets of 10 repetitions at ~70% of 1RM) and from a separate six individuals (3M, 3F) before and 24h following RE and analyzed using real‐time RT‐PCR. Collagen type I, collagen type III, and MMP‐2 mRNA levels were downregulated (p<0.05) 4h following RE, but were unchanged 24h following RE (p>0.05). The other target mRNAs remained unchanged (p>0.05) following RE. Based on these gene expression results, we speculate that acute resistance exercise stimulates both collagen type I and type III turnover in the human patellar tendon. In addition, the proteoglycans appear to be relatively refractory to a resistance exercise stimulus.

NIH R01 AG020532 (TT)
Single muscle fiber gene expression with resistance training in young women
Authors: Raue Ulrika , Conley Travis , Louis Emily , Minchev Kiril , Jemiolo Bozena , Trappe Scott
Published: June 2021

The purpose of this investigation was to examine fiber type specific gene expression (GE) before and after 12 weeks (36 sessions) of progressive resistance training (PRT) for the knee extensors. Muscle biopsies were taken from vastus lateralis of 8 young women (23 ± 2 y, 67 ± 5 kg) before and 4 h post the 1st(acute) and 36th (chronic) training session. Myogenic (MyoD, myogenin, MRF4) and proteolytic (atrogin‐1, MuRF‐1, myostatin) GE was measured in mRNA from pooled myosin heavy chain (MHC) I and IIa fibers, using real‐time RT PCR. After the 1st training session, myogenin and MRF4 GE induction (p<0.05) was targeted to the MHC I fibers, while myostatin GE decreased (p<0.05) and MuRF‐1 GE increased (p<0.05) in both fiber types. After 12 weeks of training the basal level GE of myogenin (MHC I) and MRF4 (MHC I and IIa) increased (p<0.05). In response to the 36th training bout, myogenin GE induction (p<0.05) and myostatin GE reduction (p<0.05) was targeted to the MHC IIa fibers. There was a trend (p=0.07) for MuRF‐1 to increase in both fiber types after the 36th session. In summary, these data suggest that 12 weeks of PRT alters basal level myogenic GE. Interestingly, the MHC IIa specific response for myogenin and myostatin in the chronic phase may be important for the fast‐twitch fiber adaptation that occurs with resistance training in young individuals. Supported by NIH grant AG18409.
NO‐dependent up‐regulation of antioxidant genes protects oxidative myofibers from cachectic stimuli in mice
Authors: Yan Zhen , Yu Zengli , Li Ping , Stamler Jonathan S.
Published: June 2021

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differential regulation of antioxidant gene expression accounts for the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) resulted in higher level of reactive oxygen species (ROS) and greater expression of muscle‐specific E3 ubiqitin ligases, muscle atrophy F‐box (MAFbx)/atrogin‐1 and muscle RING finger‐1 (MuRF1), in mouse glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, nitric oxide (NO) production, inducible NO synthase (iNOS) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin‐induced MAFbx/atrogin‐1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.
α‐Actinin‐3 increases concomitantly with skeletal muscle fast fibers in rat soleus muscle
Authors: Ogura Yuji , Naito Hisashi , Ichinoseki‐Sekine Noriko , Kurosaka Mitsutoshi , Kakigi Ryo , Katamoto Shizuo
Published: June 2021

α‐Actinin‐3 locates in the skeletal muscle Z‐line and forms actin‐actin crosslinks. An interesting property of α‐actinin‐3 is its expression preterm, which is restricted in fast glycolytic skeletal muscle fibers. However, little is known about the α‐actinin‐3 response to muscle fiber type transformation. Therefore, this study examined alpha‐actinin‐3 levels in soleus muscles of rats exposed to a hindlimb unloading condition, which causes a slow‐fast fiber shift in the soleus muscle. Eleven female SD rats were assigned randomly to control (CON; n = 5) or hindlimb unloading (HLU; n = 6) groups. The HLU were tail‐suspended to prevent loading of their hindlimbs for 30 days. After 30 days, soleus muscles in both groups were sampled to analyze the wet weight, myosin heavy chain (MyHC) proportion, fast myosin level, and α‐actinin‐3 protein level. The soleus wet weight of HLU was significantly lower than CON (CON; 141 ± 4 vs. HLU; 60 ± 4 mg, p<0.01). The %type II MyHC, fast myosin level, and α‐actinin‐3 level were significantly higher in HLU than in CON (8.3 ± 1.7 vs. 19.3 ± 2.7%, 100 ± 40 vs. 409 ± 77OD, 100 ± 24 vs. 831 ± 149OD, p<0.01 respectively). These results demonstrate that the α‐actinin‐3 level increases along with increases in skeletal muscle fast fibers.
Lack of skeletal muscle atrophy and myosin isoform changes in a rodent hibernator, Spermophilus lateralis, subjected to activity restriction
Authors: Rourke Bryan , Selpides Pocholo‐Jose , Choi Nicole , Roman Yanett , Jimenez Jessica
Published: June 2021

Hibernating ground squirrels demonstrate a greatly reduced, or even an absence of, atrophy in a variety of skeletal muscles and the heart during 4‐6 months of winter dormancy. We test whether these muscles are equally insensitive to inactivity induced reductions in muscle mass and changes in myosin heavy‐chain (MyHC) isoform expression. Field‐caught ground squirrels were captured during their Summer‐active periods, and subjected to cage restriction (small, individual and larger, group cages). Soleus, plantaris, medial and lateral gastrocnemius, diaphragm and cardiac muscles were studied for changes in muscle mass, MyHC isoform expression, citrate synthase activity, and atrophy related genes. Citrate synthase activity and mass was unchanged in all skeletal muscles and the heart. Cardiac alpha MyHC isoform expression was significantly increased in cage‐restricted animals. MAFbx, MuRF1, FOXO1, IGF1 and IGFBP5, and myostatin mRNA expression were also determined through RT‐PCR. The results demonstrate that activity restriction does not significantly alter skeletal muscle mass or MyHC isoform expression even in non‐hibernating ground squirrels and mechanistically suggests that protection during hibernation is an intrinsic adaptation. Funding sources: NIH SCORE, NIH RISE, CSULB.
Expression of mRNA species affecting growth and membrane homeostasis in skeletal muscle following eccentric exercise
Authors: MacNeil Lauren Gregory , Baker Steven , Tarnopolsky Mark
Published: June 2021

Eccentric exercise leads to muscle damage with our recent work suggesting an early response of components of membrane repair and remodeling at the transcriptome level. Estradiol has been reported to have membrane stabilizing properties but it is unclear if these are mediated at the transcriptome or post‐translational level. We measured skeletal muscle expression of mRNA for enzymes involved in membrane homeostasis (FOXO1, Caveolin 1 and SREBP‐2) and growth (DSCR1, DMPK and CapZα1) in healthy males following 8 days of either placebo (n=9) or 17‐beta‐estradiol (n=9) supplementation. Muscle biopsies were collected pre and post supplementation as well as 3 and 48 hours following an exercise protocol of 150 eccentric contractions. Quantification of mRNA expression was determined by RT‐PCR. FOXO1, Caveolin 1, SREBP‐2 and DSCR1 were elevated at 3h post exercise by 2.7, 1.4, 1.6 and 16.6 fold respectively, with no effect of supplementation. DMPK and CapZα1 were elevated at 48h post exercise by 2.6 and 1.8 fold respectively, with no effect of supplementation. These findings indicate an early and later temporal pattern in steady‐state mRNA content for genes involved in membrane synthesis (3h) and growth (3 and 48h) following eccentric exercise that is not influenced by17beta‐estradiol. (This research was funded by NSERC)
The disruptive respiratory effects of neurotoxic destruction of the pre‐Botzinger complex are not exacerbated during sleep
Authors: Krause K L , Forster H V , Davis S , Kiner T , Bonis J M , Qian B , Pan L G , Feroah T R
Published: June 2021

Bilateral injection of a ribosomal neurotoxin into the pre‐Botzinger complex (PBC) of rats disrupts breathing first in REM sleep, then days thereafter during NREM sleep, and eventually during wakefulness (Nat Neurosci. 9:1142–4, 2005). We test the hypothesis that incremental destruction of the PBC also leads to a state‐dependent disruption in breathing. Microtubules were bilaterally implanted into the PBC of 5 adult goats. After recovery and baseline parameters were established, increasing volumes (0.5, 1, 5, and 10uL) of ibotenic acid (IA) (50mM) were injected into the PBC, with one week between injections. Goats were studied for 5 hours post injection (accompanying abstract) and each night 9 to 14 hours after injection. Minutes after each injection, breathing frequency increased and eupneic airflow and respiratory muscle pattern were disrupted. Then, 9–14 hours post injection, these disruptions were most pronounced during the awake state, and less during NREM and REM sleep. One week after the 10uL injection, breathing pattern and blood gases did not differ between state, or from pre‐IA control. Histological analysis showed >85% destruction of the PBC. Thus, after incremental destruction of the PBC, plasticity within the respiratory network provides a respiratory rhythm appropriate to sustain homeostasis during awake, NREM, and REM states. (Supported by the Department of Veterans Affairs and NIH HL25739)
Blockade of GABA and glycine reuptake differentially affects temporal and spectral characteristics of phrenic nerve discharge in arterially‐perfused adult rat
Authors: Chen Xinnian , Chon Ki H. , Solomon Irene C.
Published: June 2021

Fast synaptic inhibition plays an essential role in control of the central respiratory network. Recently, we have shown that blockade of GABAA and glycine receptors in an arterially‐perfused adult rat preparation alters the temporal and spectral characteristics of phrenic nerve discharge and markedly reduces inspiratory network complexity. The influence of excess endogenous GABA and glycine on phrenic motor output, however, is not known. Therefore, we examined the effects of blockade of GABA and glycine reuptake using sarcosine (SAR; n=6) and nipecotic acid (NA; n=5), respectively, on phrenic nerve discharge in the same preparation. Perfusion with either reuptake blocker (100 mM) ultimately abolished phrenic nerve discharge; the effects of SAR resulted from a decrease in burst amplitude (P=0.0001) while those of NA resulted from a decrease in burst frequency (P=0.004). SAR and NA slightly enhanced MFO spectral power; however, HFO spectral power was differentially affected (i.e., SAR decreased; NA increased). Inspiratory network complexity (approximate entropy) was unchanged by SAR or NA. These data demonstrate that excess endogenous GABA and glycine differentially modulate the temporal and spectral characteristics of phrenic nerve discharge, providing additional evidence supporting separate and distinct roles for GABA and glycine in central respiratory neural control. Support: NS045321, NS049310
Episodic breathing patterns in neonatal rats: neurochemical modulation, developmental changes and pontine influences
Authors: Fong Angelina Yin , Milsom William K
Published: June 2021

Episodic breathing is prevalent in premature infants and is found in fetal rats but rarely reported in postnatal (PN) in vitro preparations. We observed episodic breathing patterns in the in vitro pontomedullary‐spinal cord preparation from rat pups on day of birth (P0) and PN day 2 (P2), but the occurrence declined with PN age with 70% of P0 and 20% of P2 preparations exhibited episodic patterns. In P0 preparations, episodic inspiratory bursting was recorded from cervical rootlets (C4/C1), hypoglossal and thoracic nerves (T1/T2), and episodic expiratory bursts coupled to inspiratory bursts were recorded from the 12th thoracic nerve rootlet. As neonates are highly sensitive to respiratory depression by opioids, we hypothesized that endogenous opioids participate in the production of the apneas between the episodes. Activation of opioid receptors (DAMGO 1–2μM) increased apnea duration but antagonism with naloxone (1–5μM) did not alter apnea duration or the episodic pattern, suggesting that endogenous opioids do not mediate the episodic rhythm. We also examined the role of the pons in the generation of the episodic rhythm. Both chemical inhibition and physical removal of the pons eliminated the episodic breathing pattern. Our data suggests that the pons is involved in the generation of episodic rhythms but that this involvement decreases and is lost over the first two PN days.
Maturation Influences effects of long‐term facilitation induced changes in inspiratory network complexity in urethane‐anesthetized neonatal rats
Authors: Reid Inefta M , Solomon Irene C
Published: June 2021

Long‐term facilitation (LTF) is a form of respiratory plasticity, which is expressed as a persistent increase in inspiratory motor output following intermittent, but not continuous, hypoxia. Although numerous studies have examined timing and magnitude changes in respiratory LTF, changes in inspiratory network complexity (quantified by approximate entropy ApEn analysis) in neonatal Sprague‐Dawley rats have only begun to be explored. Here we extend earlier analyses by examining ApEn of diaphragm EMG bursts recorded from spontaneously breathing urethane‐anesthetized neonatal rats (P12–21 n=18) before and at 5‐min intervals for at least 60‐min following three 5‐min episodes of hypoxia (8% O2); all rats were continuously supplied with 4% CO2 to minimize hypoxic respiratory depression. Under these conditions, respiratory LTF was seen mainly as an increase in burst frequency although in some cases an increase in amplitude was observed. During LTF, ApEn values increased from 0.37±0.04 to 0.42±0.02 (P=0.004) P12–P13 rats(n=7) and from 0.40±0.03 to 0.43±0.03 (P=0.04) in P21 rats(n=3), with the peak response seen at ≥50 min. In contrast, ApEn values decreased from 0.40±0.03 to 0.36±0.03 (P=0.007) in P14–P18 rats(n=8), with the peak response seen at ≤45 min. These data show that inspiratory network complexity is modified during LTF, and that the stage of development affects LTF‐related network reconfiguration.
Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow
Authors: Weinstein Alan M. , Sontag Eduardo D.
Published: June 2021

Normally, there are wide swings in proximal tubule fluid flow and proportional changes in Na reabsorption across tubule epithelial cells. This “glomerulotubular balance” occurs in the absence of substantial change in cell volume, and is a challenge to coordinate luminal solute entry with peritubular solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A model of rat proximal tubule epithelium is linearized about a reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three variables: cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure stabilizes Na flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na flux varies over a factor of two. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. The increase in Na flux is achieved with upregulation of luminal Na/H exchange and Na‐glucose cotransport, with increased peritubular Na‐3HCO3 and K‐Cl cotransport, and with increased Na,K‐ATPase activity. The configuration of activated transporters emerges as testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent cytoskeletal effects of luminal flow. Supported by NIH RO1‐DK‐29857.
Computational modelling identifies impact of subtle anatomical variation on skeletal muscle local calcium dynamics
Authors: Groenendaal Willemijn , Riel Natal van , Jeneson Jeroen , Eikelder Huub ten , Nicolay Klaas , Hilbers Peter
Published: June 2021

Calcium is the main regulator of skeletal muscle metabolic activity. The question has been addressed whether the highly structured spatial organization of sites of Ca2+ release, uptake and action in skeletal muscle substantially impacts the dynamics of cytosolic Ca2+ handling and thereby the physiology of the cell. Hereto, the spatiotemporal dynamics of the free calcium distribution in a fast‐twitch muscle sarcomere was studied using a reaction‐diffusion computational model.

The model was based on the model of Baylor and Hollingworth (J Gen Physiol. 1998 112:297–316), but was adapted to handle local calcium dynamics in mouse EDL fast twitch muscle at 35C. Furthermore, the Ca2+ mass balance was closed by adding a mathematical representation of the sarcoplasmic reticulum. Experimental calcium time courses (high time resolution, but spatially averaged) obtained under physiological conditions (35C, 125 Hz stimulation frequency) were used for model validation.

The model showed that subtle changes in sarcomere microstructure influenced the local calcium concentration. Furthermore, local calcium concentration sensed by mitochondria was higher than average calcium concentration and also above the activation constant of the mitochondria, whereas the local concentration was not. Furthermore, the free Ca2+ concentration was higher at the positions with troponin C than without troponin C.
Heart rate as a measure of energy expenditure in a small rodent – the golden spiny mouse (Acomys russatus)
Authors: Choshniak Itzhak , Shargal Eyal , Brosh Arieh
Published: June 2021

While monitoring heart rate (fH) as a measure of energy expenditure is a widely‐used approach in large animals, much uncertainty remains in the interpretation of results using the same approach in small rodents. In the present study, the validity of fH monitoring as an estimate of oxygen consumption rate (VO2) was tested in caged golden spiny mice under controlled conditions. Four mature males (72.5 ± 8.8 g) were surgically implanted with small transmitters (3.5g, Data Sciences International), enabling the continuous recording of heart rate, core body temperature and activity. Measurements of VO2 were taken at 72 hour intervals. Animals were maintained under controlled conditions (30 ± 1°C, 12L‐12D), and fed either ad lib. or restricted to 50% of their ad lib. food intake. Significant daily patterns of fH and VO2 (P<0.001) were observed in animals under both feeding regimes. The daily pattern was affected mainly by time of feeding, particularly in the food‐restricted animals. Heart rate was positively related to VO2 both in mice fed ad lib. (P<0.001, R2= 0.918), and in food‐restricted mice (P<0.001, R2= 0.94). Within and between animal oxygen pulse (O2P) varied during the day and between days independently of feeding regime. The data suggest that despite the variability in O2P observed between and within animals, fH monitoring offers a relatively reliable indicator for prediction of group means VO2 in resting spiny mice.
Enzyme activities in mammals adapted to extreme environments: hibernators and divers versus terrestrial mammals
Authors: Pearson Linnea E. , Gullett Keyona C. , Florant Gregory L. , Kanatous Shane B.
Published: June 2021

The objective of this study was to examine the metabolic plasticity of skeletal and cardiac muscle when exposed to varying temperatures. When faced with extreme temperature variation, activities of metabolic enzymes in homeothermic terrestrial mammals are directly correlated with temperature. We investigated the physiological adaptations in two extreme mammalian conditions; a diving mammal, a homeotherm, able to maintain function at low temperature and a hibernator, a heterotherm that decreases activity in response to a decrease in temperature. We measured the enzyme activity of citrate synthase, lactate dehydrogenase and catalase at 10, 25, 37 and 40°C in cardiac and skeletal muscle of the golden mantle ground squirrel (hibernator), harbor seal (diving mammal) and rat (terrestrial mammal). Surprisingly, our results indicate there is no direct correlation between temperature and enzymatic activity in the homeothermic diving mammal. In addition, the enzyme activity of the diving mammal showed a similar pattern of activity in response to temperature variation as the ground squirrel. And as expected, due to its smaller size, the ground squirrel had greater overall enzyme activity. Our results suggest there are similar adaptations in the cardiac and skeletal muscles of diving and hibernating mammals under varying temperatures that preserve enzyme function even at low environmental temperatures.
Gait disturbances in BIO TO‐2 hamster model for muscular dystrophy
Authors: Hampton Thomas G. , Kale Ajit , Amende Ivo , McCue Scott , Bhagavan Hemmi N. , VanDongen Case G.
Published: June 2021

BIO TO2 hamster that has a deletion in the delta sarcoglycan gene is an excellent animal model for muscular dystrophy. Gait disturbances, important clinically in patients with muscular dystrophy, have not yet been described in BIO TO2 hamsters. We therefore compared the gait of BIO TO2 dystrophic hamsters with healthy BIO F1B control hamsters using ventral plane videography. Some hamsters were provided access to a voluntary activity wheel for one month prior to study to test whether exercise would affect disease severity. When BIO TO2 and BIO F1B hamsters (12 each) were made to walk on a transparent treadmill belt, we found kinematic and postural changes in BIO TO2 hamsters including significantly shorter swing, stride and stance durations. Stride length was ~13% shorter in BIO TO2 hamsters at 3 and 9 mo. Propulsion duration of the hind limbs, an indicator of muscle strength, was shorter in 9 mo old BIO TO2 hamsters. This deficit was also apparent at 3 mo. Braking duration, reflecting generation of ground reaction forces, was delayed in 9 mo old BIO TO2 hamsters. Hind paw eversion, evidence of muscle weakness, was greater in 9 mo old BIO TO2. The gait disturbances were absent in 3 mo old BIO TO2 hamsters that had access to voluntary wheels, and the stride lengths were comparable to 3 mo old BIO F1B hamsters that had activity wheels. However, comparable gait disturbances were observed in 9 mo old dystrophic hamsters that were provided voluntary wheels for one month. Our findings show that the dystrophic BIO TO2 hamsters recapitulate the functional features of human muscular dystrophy as early as 3 mo of age. Exercise has some benefit in delaying the onset of disease symptoms. Early detection and quantitative measures of gait abnormalities will accelerate the development of new drugs for treating muscular dystrophy.
Effects of oxidants on global phosphatase activity in isolated diaphragm
Authors: Clanton Thomas L , Wright Valerie P
Published: June 2021

Phosphatases are potential cell redox sensors. We hypothesize that skeletal muscle phosphatase (PPase) activity is reduced by exposure to low levels of oxidants. Rat diaphragm bundles were treated with H2O2 (5, 50, 500 μM) or 2,3‐dimethoxy‐1‐naphthoquinone (DMNQ; 1,10, or 100 μM; an intracellular O2•− generator). Supernatants were assayed for oxidized proteins, total PPase, serine/threonine PPase (ser/thr‐PPase) or tyrosine PPase (tyr‐PPase) activities. Total PPase activity was significantly reduced at all H2O2 doses, (20% at 500 μM) whereas Ser/thr‐PPase activity was significantly reduced at 50 and 500 μM H2O2. Total PPase and ser/thr‐PPase activities were significantly reduced at 10μM and 100μM DMNQ, whereas Tyr‐PPase activity decreased significantly at all doses. DTT treatment of supernatants increased all PPase activities but surprisingly did not reverse the dose‐dependent effects of oxidants. Assays of global protein oxidation displayed mild carbonyl formation at 500μM H2O2 but there were no other indications of protein oxidation. The data are consistent with global phosphatase activity being highly sensitive to local cellular redox changes within a near physiologic range. NIH 53333
Recovery from Oxidative Stress: Effects on the Tight Junction in Epithelial Cells.
Authors: Arthur D'Ann E. , Gonzalez Jeannette E. , King Jonathan M.
Published: June 2021

Loss of barrier function in kidney epithelial cells due to alterations in the tight junction (TJ) may be a contributory mechanism of ischemia‐reperfusion injury. The objective of this study was to demonstrate the changes in TJ function and protein composition during recovery after exposure to oxidative stress. Oxidative stress was induced in MDCK Type II cells by exposure to H2O2, and cells were subsequently recovered for 24 hours. Transepithelial electrical resistance (TER) and solute flux were used as measures of barrier function. Protein expression and localization was determined for TJ proteins during the recovery period. Moderate doses of H2O2 perturbed TJ function without exhibiting a significant cytotoxic effect. ERK‐1/2, an enzyme of the mitogen activated protein kinase (MAPK) pathway, was activated within minutes of exposure to H2O2. Exposure to H2O2 with subsequent recovery resulted in initially decreased TER followed by recovery of function and was accompanied by changes in expression and localization of the TJ proteins occludin and claudin‐1. Inhibition of ERK activation significantly attenuated recovery changes in TER and TJ protein composition induced by H2O2. Our findings suggest that the TJ in kidney epithelial cells is altered by H2O2 exposure and recovery. These changes appear to be mediated by activation of the MAPK pathway. This work was supported by NIH Grant DK065652 and the HHMI Foundation.
Heat Shock Protein 27 (Hsp27) Protects Lung Epithelial Cells from Hyperoxia‐Induced Cell Death
Authors: Xu Dong , Truog William E , Perez Ricardo E , Ekekezie Ike I , Gerthoffer William T , Shao Lei
Published: June 2021

Supplemental oxygen therapy (hyperoxia) is a common life‐saving practice for preterm babies with respiratory distress. Prolonged hyperoxia is one of the major contributing factors in cell death and the development of bronchopulmonary dysplasia (BPD), a chronic lung disease, in preterm babies. Hsp27 is the smallest inducible heat shock chaperone protein that modulates the ability of cells to respond to oxidative stress. Our DNA Superarray analysis revealed that hyperoxia significantly decreased Hsp27 gene expression in newborn rat lungs. Immunohistochemistry of Hsp27 confirmed that Hsp27 protein in epithelial cells of neonatal rat lungs was markedly decreased after hyperoxic exposure. In cultured human lung epithelial cells (A549), prolonged hyperoxia significantly reduced Hsp27 expression and caused cell death. Overexpression of hHsp27 could prevent hyperoxia‐induced cell death in cultured human lung epithelial cells. Our studies suggest that hyperoxia reduces Hsp27 expression in lung epithelial cells and overexpression of hHsp27 confers cytoprotection against hyperoxia‐induced cell death in lung epithelial cells.
The Effect of Superoxide Generation by Ascorbic Acid and Imidazole on Coronary and Pulmonary Arteries
Authors: Sylvester Francis Albert , Hall Jeffrey , Hallack April , Kipp Brian H.
Published: June 2021

The purpose of this study was to evaluate the responses of coronary and pulmonary arteries to the generation of superoxide (O2.) via organic redox complex formation between ascorbic acid and imidazole. Imidazole facilitates the transfer of electrons from ascorbic acid to O2 resulting in the generation of O2. Using a Clark style electrode, oxygen levels were monitored in solutions of ascorbic acid and imidazole and decreasing oxygen levels were used as an indication of O2. generation. In another series of experiments, small rings of porcine coronary and pulmonary arteries were dissected, mounted in isolated organ baths, and administered increasing concentrations (10−8–10−3 M) of ascorbic acid, glycine, imidazole, or ascorbic acid and imidazole. Vascular responses were recorded as changes in tension. The combination of ascorbic acid and imidazole, but not ascorbic acid or glycine, caused a significant vasoconstriction in both coronary and pulmonary arteries. Imidazole induced vasoconstriction in coronary arteries. Taken together, these results suggest that ascorbic acid in the presence of imidazole may increase O2. production resulting in an increase in tension in coronary and pulmonary arteries. This research was supported by a Grand Valley State University Research Grant‐in‐Aid.
Bilirubin oxidation products (BOXes) are produced by biochemical oxidation involving free peroxynitrite
Authors: Wurster William L. , Loftspring M. , Pyne‐Geithman G. J. , Clark J. F.
Published: June 2021

Bilirubin oxidation products (BOXes), 1‐(1,5‐dihydropyrrole‐2‐ylidene)acetamide are vasoactive molecules with clinical importance. They are found in the cerebrospinal fluid of patients who have had hemorrhagic strokes. These compounds exhibit biological activity. We report here the production of BOXes via oxidation of unconjugated bilirubin (UBR) by peroxynitrite (PN) under conditions approximating in vivo conditions. Using a mixture of PN, buffer, UBR and O2 we have generated BOXes, confirmed by UV/Vis consistent with known BOXes mixtures, and by HPLC‐MS. The oxidation kinetics is rapid at pH 7.5. Simple agonists and antagonists of the reaction exist. This is the second report of BOXes production using a well‐defined biological reaction. Considering the complexity of the process of maintaining vascular tone, it is not surprising that multiple routes to vasospastic compounds may exist. Following subarachnoid hemorrhage the amounts of UBR in the spinal fluid and chemical species, such as PN, created by oxidative stress are undoubtedly present. The oxidation of UBR to yield bioactive molecules, such as BOXes, is an important discovery, which expands the role of UBR in pathological processes. Therefore, in vitro production of BOXes could imply a specific in vivo therapeutic target for preventing BOXes production and thereby treating or preventing cerebral vasospasm. NIH: NS050569.
IL‐10 mitigates TNF‐alpha induced cardiomyocyte apoptosis: Role of mitogen‐activated protein kinases
Authors: Dhingra Sanjiv , Sharma Anita K , Bajaj Anju , Singal Pawan K
Published: June 2021

Our recent data suggested that IL‐10 modulates the TNF‐[alpha] induced oxidative stress in cardiomyocytes. However, intracellular signaling events responsible for this effect are not known. Present study investigates the role of p38 and ERK 1/2 MAP kinases in TNF‐[alpha] induced cardiomyocyte apoptosis and its modulation by IL‐10. Cardiomyocytes isolated from Sprague Dawley rats were exposed to TNF‐[alpha] (10ng/ml), IL‐10 (10ng/ml) and IL‐10+TNF‐[alpha] (ratio 1) for 4hrs. H2O2 (positive control) and antioxidant trolox (20 [mu]mol/L) were used to confirm the role of oxidative stress. H2O2 (100 [mu]Ì) increased oxidative stress and apoptosis, TNF‐[alpha] mimicked these effects. Exposure to TNF‐[alpha]increased ROS production, caused cell injury, increased the number of apoptotic cells and Bax/Bcl‐xl ratio. This change was associated with an increase in p38 and a decrease in ERK 1/2 phosphorylation. IL‐10 by itself had no effect on these MAP kinases, but it prevented TNF‐[alpha] induced changes. Trolox mitigated TNF‐[alpha] induced changes. Pre‐exposure of cells to p38 inhibitor SB‐203580, prevented TNF‐[alpha] induced changes. Inhibition of ERK pathway with PD98059 attenuated protective role of IL‐10 against TNF‐[alpha] induced apoptosis. Present data suggest the essential role of p38 and ERK 1/2 in TNF‐[alpha] induced cardiomyocyte apoptosis and its modulation by IL‐10 (supported by CIHR).
Effects of in ovo exposure to hyperoxia on chicken ductus arteriosus reactivity
Authors: Villamor Eduardo , van der Sterren Saskia , Cogolludo Angel L
Published: June 2021

In vivo exposure to hyperoxic environment modifies the in vitro vasomotor response of pulmonary and systemic vessels. We hypothesized that prenatal exposure to hyperoxia would alter the development of ductus arteriosus (DA) reactivity. Hyperoxia was induced by incubating fertilized eggs at 60% O2 from day 15 to 19 of the 21‐d incubation period. DA contractility was studied on day 19 (non‐internally‐pipped embryos) Relaxant responses were studied after precontraction with 125 mM KCl. Hyperoxia did not affect embryonic survival or growth. The contractions induced by 21% O2, KCl, the inhibitor of KV channels 4‐aminopyridine, phenylephrine, and endothelin‐1 and the relaxations induced by acetylcholine, sodium nitroprusside, forskolin, isoproterenol, and the Rho kinase inhibitor OH‐fasudil were similar in DA from embryos incubated under normoxic or hyperoxic conditions. In contrast, the membrane permeable hydroperoxide t‐butyl‐hydroperoxide (1 μM–0.1mM) induced concentration‐dependent contraction in the DA from normoxic embryos and relaxation in the DA from hyperoxic embryos. Reactive oxygen species are complex signalling molecule that produce relaxation in some vessels and contraction in others. Our results indicate that exposure to oxidant stress is capable of modifying the pattern of response of the DA to hydrogen peroxide.

Supported by “Fundación de Investigación Médica Mutua Madrileña”
Hypotaurine protects against H2S‐induced oxidative stress and cell death in erythrocytes from an H2S‐tolerant marine worm
Authors: Ortega Jessica A , Ortega Jocelynn M , Julian David
Published: June 2021

Many marine invertebrates adapted to H2S contain high concentrations of the organic osmolyte hypotaurine (H). In the presence of H2S, H is converted to thiotaurine (Th), and the Th:(H+Th) ratio is higher in animals from environments with higher H2S, suggesting that H can serve as a H2S buffer. However, H and possibly Th are antioxidants and therefore may also protect against free radicals produced by H2S oxidation. We used erythrocytes from the H2S‐tolerant marine polychaete Glycera dibranchiata to determine whether H reduces cell death and oxidative stress caused by H2S exposure in vitro. Greater than 90% of cells were killed by exposure to 0.3% H2S gas (based on calcein‐AM staining) but this death was decreased by 60%, 78%, and 83% with 0.5, 5.0 and 50 mM H, respectively (p<0.001 for each, n=8). Similar trends were observed at both higher and lower H2S concentrations (0.1, 1 and 3% H2S). Oxidative stress (based on H2DCFDA) was increased 67% by H2S (p<0.0001), but this increase was attenuated by H (p=0.0002). In contrast, H did not decrease the effect of H2O2 (0.01–10 mM) on cell death (p=0.001) or oxidative stress (p=0.038), and H that was pre‐converted to Th did not reduce H2S‐induced cell death (p<0.0001). Therefore, hypotaurine protects against cell death and oxidative stress, although it remains unknown whether these effects are due to sulfide buffering or free radical scavenging.