bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammation Increases the Excitatory Synaptic Transmission in the Nucleus Tractus Solitarius (NTS) of Rats Submitted to Sustained Hypoxia
Authors: Silveira Ludmila Lima , Accorsi‐Mendonca Daniela , Bonagamba Leni GH , Machado Benedito H.
Published: November 2023

Short‐term sustained hypoxia (SH) is associated with cardiorespiratory changes and inflammatory process in the central nervous system. SH also promotes changes in excitatory synaptic transmission in the NTS. In this study we evaluated the possible role of inflammation triggered by SH on the excitatory synaptic transmission of NTS neurons sending projections to the Ventral Medulla (VM). For this purpose, we used juvenile Wistar rats (90–120g), which were divided into 4 groups: 1) control (FiO2=20.8%) treated with vehicle (saline + distilled water, i.p), 2) control treated with the anti‐inflammatory Minocycline (30mg/kg, i.p), 3) SH (FiO2=10%) treated with vehicle, 4) SH treated with Minocycline. The rats were treated for 3 days and then exposed to SH or normoxia protocol for 24 h. At the end of these protocols, the animals were decapitated and brainstem slices were obtained. Whole cell patch‐clamp technique was used to electrophysiological recordings of NTS neurons sending projections to the VM which were previously identified by retrograde labeling. SH produced no changes in the intrinsic properties of the NTS neurons, but spontaneous excitatory postsynaptic currents (sEPSCs) of NTS neurons from SH animals treated with vehicle presented a higher frequency when compared with the group that was maintained in normoxic conditions [2,4±0,4 (n=7) vs 0,96±0.2Hz (n=13)]. This increase that was not observed in the SH group treated with Minocycline when compared to normoxic group [0,9±0.3Hz (n=6) vs 0,9±0.2Hz (n=7), two‐way ANOVA followed by Bonferroni's post‐test]. The data show that inflammation induces by SH contributes to the increase in excitatory synaptic transmission in the NTS neurons sending projections to VM.

Support or Funding Information

Financial support: CAPES, CNPq, FAPESP.
Brain‐Derived Neurotrophic Factor in Nucleus Tractus Solitarius is increased in Pressure Overload‐Induced Cardiac Hypertrophy via Heart‐to‐Brain Axis
Authors: Takenaka Risa , Ito Koji , Kishi Takuya , Hirooka Yoshitaka
Published: November 2023

Introduction

Cardiac hypertrophy is an independent risk factor for heart failure, and result in baroreflex dysfunction. Baroreflex dysfunction further deteriorates cardiac hypertrophy and, if prolonged, may progress into heart failure with a vicious cycle. However, it remains unclear how cardiac hypertrophy worsens baroreflex via the heart‐to‐brain axis. We focused cardiac afferent nerve as a component of the heart‐to‐brain axis. Nucleus tractus solitarius (NTS) is primary nucleus in central baroreceptor circuit and received cardiac afferent nerve. It is suggested that increased brain‐derived neurotrophic factor (BDNF) in NTS reduces the neuronal activity, which might disrupt baroreflex. The cardiac afferent nerve terminals express a transient receptor potential vanilloid type 1 (TRPV1), and TRPV1 increases in the hypertrophic heart from transgenic mice. We hypothesized that pressure overload‐induced cardiac hypertrophy increases BDNF in NTS via TRPV1 in cardiac afferent nerve.

Methods

We divided wild‐type (WT) and TRPV1 knockout mice (KO) with aortic banding (AB) or sham operation. At 8 weeks after the operation, we evaluated cardiac function by echocardiography and BDNF levels in the NTS by Western blot analysis.

Result

Echocardiographic left ventricular hypertrophy and heart weight were significantly greater in WT‐AB than in WT‐sham, and were attenuated in KO‐AB. Western blot analysis revealed a significant increase in BDNF in NTS of WT‐AB compared with WT‐sham (1.26±0.01 vs. 0.90±0.02 units, n=4, P<0.05). However, KO‐AB had significantly lower BDNF in NTS than WT‐AB (1.26±0.01 vs. 1.02±0.02 units, n=4, P<0.05).

Conclusion

Increased BDNF in NTS by pressure overload‐induced cardiac hypertrophy was inhibited by knockout of TRPV1. These findings support the novel concept that cardiac hypertrophy disrupts baroreflex via the “heart (TRPV1)‐to‐brain (BDNF in NTS) axis”.

Support or Funding Information

This study was supported by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24390198 to Dr. Hirooka and 25461112 to Dr. Ito).
Select Inhibition of Arcuate NPY/AgRP Neurons Increases Arterial Pressure and Sympathetic Nerve Activity in Mice
Authors: Shi Zhigang , Brooks Virginia L.
Published: November 2023

Using designer receptors exclusively activated by designer drugs (DREADD) technology, we recently demonstrated that select activation of arcuate nucleus (ArcN) neurons that express Neuropeptide Y (NPY) and agouti‐related protein (AgRP) rapidly and profoundly decreases splanchnic sympathetic nerve activity (SSNA) and mean arterial pressure (MAP) in male mice. The present study tested the hypothesis that ArcN NPY/AgRP neurons tonically suppress SSNA, by determining if inhibition of these neurons increases SSNA. To selectively inhibit ArcN NPY/AgRP neurons, the mutated inhibitory muscarinic receptor (hM4Di) was specifically expressed via bilateral nanoinjection of Cre‐recombinase‐dependent adeno‐associated virus (AAV)‐hM4Di‐mCherry into the ArcN of AgRP‐Ires‐cre mice. After >2 weeks recovery, mice were anesthetized with α‐chloralose and received ip injections of the otherwise inert ligand Clozapine‐N‐oxide (CNO; 0.3 mg/kg; n=4), to activate hM4Di, or saline (n=3). CNO slowly (after ~45 min) increased SSNA from 99±1 to 151±3 % control and increased MAP from 76±7 to 84±7 mmHg (all P<0.01, ANOVA); however, saline had no effects (SSNA: 101±1 to 95±5 % control; MAP: 89±6 to 88±6 mmHg). ArcN NPY/AgRP neurons send major projections to the hypothalamic paraventricular nucleus (PVN) and the dorsal medial hypothalamus (DMH). To begin to identify the sites at which ArcN NPY/AgRP activation (or inhibition) alters SSNA/MAP, we next nanoinjected (30 nL) NPY (3 pmol) or the NPY Y1 receptor antagonist BIBO3304 (0.3 nmol) into the PVN or DMH in anesthetized wild type mice. PVN NPY decreased (n=4; P<0.05) MAP (92±4 to 84±5 mmHg) and SSNA (99±1 to 64±5 % control), whereas PVN BIBO3304 increased (n=3; P<0.05) MAP (82±7 to 95±5 mmHg) and SSNA (95±3 to 137±9 % control). Similarly, DMH NPY decreased (n=3; P<0.05) MAP (99±7 to 79±3 mmHg) and SSNA (100±1 to 71±4 % control), and DMH BIBO3304 increased (n=3; P<0.05) MAP (92±3 to 106±4 mmHg) and SSNA (101±6 to 141±9 % control). In conclusion, ArcN NPY/AgRP neurons tonically suppress SSNA and MAP, possibly in part via NPY projections to the PVN or DMH.

Support or Funding Information

AHA 12GRNT11550018 and 15POST23040042
Oxidative Stress Contributes to Microvascular Endothelial Dysfunction in Humans with Major Depressive Disorder
Authors: Greaney Jody L , Santhanam Lakshmi , Saunders Erika F , Alexander Lacy M
Published: November 2023

Vascular dysfunction is evident in humans with major depressive disorder (MDD); however, the specific molecular mediators remain unclear. In rodent models of depression, oxidative stress‐induced reductions in nitric oxide (NO) bioavailability contribute to impaired endothelium‐dependent vasodilation. We hypothesized that 1) markers of oxidant stress expression and activity would be increased in adults with MDD compared to healthy control (HC) subjects (in vitro), 2) NO‐dependent vasodilation would be attenuated in adults with MDD (in vivo), and 3) acute in vivo scavenging of reactive oxygen species (ROS) would improve endothelial function in MDD. 6 HC (36±9 yrs) and 5 adults with MDD (33±8 yrs) participated; MDD subjects had clinically significant depressive symptoms, classified according to the Center for Epidemiologic Studies Depression Scale (CES‐D score 34±3). NADPH oxidase expression (p47phox subunit; Western blot analysis) and total ROS production (fluorometric assay) were quantified in forearm cutaneous homogenates. Red blood cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis infusion of acetylcholine alone (ACh; 10−8‐10−1M) and during co‐infusion with the non‐specific NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME; 20mM) and the non‐specific antioxidant ascorbate (AA; 20mM). Cutaneous vascular conductance (CVC=flux/mean arterial pressure) was expressed as a percentage of maximum (sodium nitroprusside; local heat to 43°C). NADPH oxidase expression and total ROS production were increased in adults with MDD (P<0.05 for both). ACh‐induced vasodilation was blunted in adults with MDD (96±2 HC v. 72±5 MDD %CVCmax; P<0.05); however, there were no group differences in cutaneous vasodilation during NO synthase inhibition (46±4 HC v. 49±6 MDD %CVCmax; P=0.7). AA infusion improved ACh‐induced vasodilation in MDD (72±5 ACh v. 89±10 AA %CVCmax; P=0.11). These data demonstrate impaired NO‐mediated endothelium‐dependent vasodilation in MDD and further suggest a biochemical mechanism and functional role for oxidant stress in mediating vascular dysfunction in MDD.

Support or Funding Information

HL120471‐03 and HL093238‐06
Effects of High Fat Diet and Exercise Intervention Before and During Pregnancy on the Hearts of C57BL/6 Mothers
Authors: Grue Katherine A. , Kaur Gurvinder , Joiner Hayli E. , Jacobo A. Unique , Ramalingam Latha , Shen Chwan‐Li , Dufour Jannette M. , Moustaid‐Moussa Naima , Chung Eunhee
Published: November 2023

Obesity is associated with pathological cardiac hypertrophy, while pregnancy and exercise induce physiological cardiac hypertrophy. Unlike physiological growth, pathological cardiac hypertrophy is associated with a re‐expression of fetal genes that are commonly expressed in fetal heart and alteration of cardiac metabolism. Currently, the interactive effects of these stimuli on cardiac adaptation are not well understood. Thus, this study aims to test the hypothesis that exercise before and during pregnancy would alleviate high fat diet induced cardiac maladaptation. Using C57BL/6 virgin female mice as a model, we fed the animals either a high fat diet (45% kcal fat) or a low fat diet (LFD: 10% kcal fat) for twelve weeks. Four weeks prior to pregnancy initiation, the HFD group mice were split into either sedentary (HFD) or exercised mice which participated in voluntary wheel running throughout gestation (HFD+Ex). The study was terminated at gestation day 19. Exercise during pregnancy did not improve HFD induced insulin resistance at day 15 of gestation. However, serum insulin levels at gestation day 19 were significantly reduced in HFD+Ex compared to the HFD suggesting better insulin sensitivity. Re‐expression of fetal genes were not observed in any group. However, hearts from HFD+Ex exhibited significant upregulation of genes regulating lipid and glucose metabolism (medium‐chain acyl‐coA dehydrogenase, carnitine palmitoyltransferase I, insulin receptor substrate 1, and hexokinase 1), and mitochondrial biogenesis (Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha) compared to HFD, but not the LFD. These data suggest that while exercise before and during pregnancy did not alter high fat diet associated whole body insulin resistance, it upregulated the metabolic genes of the heart of pregnant mothers. Our results suggest that exercise may be beneficial for cardiac metabolic health by circumventing high‐fat diet induced fatty acid accumulation in the heart.
Weight supported quadrupedal treadmill exercise improves insulin sensitivity in a rat model of spinal cord injury and diabetes
Authors: Traughber Terence , Ruiz Emanuel , Estrada Christina , Hinahon Erika , Villanueva Silvia , Deriquito Sara , Yamazaki Katrina , de Leon Ray
Published: November 2023

Previous studies have shown exercise is a simple and economical therapeutic modality that effectively benefits patients with type 2 diabetes. Spinal cord injured (SCI) individuals have a high risk for developing type 2 diabetes. However, there have been no studies that have examined the effects of treadmill exercise in individuals with SCI and type 2 diabetes. We examined the effects of imposing quadrupedal body weight supported treadmill exercise in a rodent model of SCI and type 2 diabetes. Seventeen rats received a severe spinal cord contusion (T10). One day after the contusion, the rats were placed on a high energy diet (10% fat, 20% sucrose, and 70% rodent chow) and 40 days later, a low dose of streptozotocin (STZ; 30 mg/kg, i.p.) was administered. The rats were assigned to an exercise (n=8) or non‐exercise group (n=9). Exercise consisted of weight supported, quadrupedal treadmill walking (23 cm/s) for 1 hr/day, 5 days/week for 6 weeks. Insulin tolerance tests were performed, hemodynamic measurements were recorded, and heart and hindlimb muscle tissue was harvested after the rats were killed. Based on fasting blood glucose, the high energy diet and STZ treatment was successful in inducing diabetes in the rats (i.e. >140 mg/dL; exercise: 202±50 mg/dL; non‐exercise: 146±29 mg/dL). Fasting blood glucose levels were not significantly different between the groups. However, insulin sensitivity was improved by exercise based on insulin tolerance test. We found significant differences in glucose levels 45 and 60 min after insulin injection indicating a greater insulin sensitivity in the exercised group. Exercise also significantly increased tibialis anterior, gastrocnemius and soleus muscle weights by 7%, 20%, 31% respectively indicating the exercise was effective in activating hindlimb activity in the SCI rats. These findings suggest for the first time that weight supported treadmill exercise can reduce metabolic abnormalities that occur after SCI.

Support or Funding Information

NIH grant R15NS082711 to R. de Leon
Disruption of Adenylyl Cyclase Type 5 Mimics Exercise Training
Authors: Zhang Jie , Keim Catharine , Lai Lo , Yan Lin , Vatner Stephen F. , Vatner Dorothy E
Published: November 2023

Adenylyl cyclase type 5 knockout mice (AC5 KO) exhibit enhanced exercise compared to their wild type (WT) littermates and also have enhanced mitochondrial function. Enhanced exercise performance, mediated in part by increased mitochondrial function, is also a feature of exercise training. Accordingly, the goal of this investigation was to compare mechanisms mediating the improved exercise in AC5 KO with that of exercise training in their WT littermates. To accomplish this, we compared genes up or down regulated in the two models using deep sequencing techniques. We also examined the phenotype of the skeletal muscle of AC5 KO to determine if it resembled exercise trained skeletal muscle. Exercise performance, measured by maximal distance, was increased in AC5 KO (n=6) by 32±9%, p<0.01, above levels in WT (449±39m, n=7). Similar significant increases were observed in maximal speed and work to exhaustion in AC5 KO. Chronic exercise training (1 hour of exercise training every day for one month) increased performance in WT (n=7), e.g., distance rose to 569±14m, similar to levels in AC5 KO without training. Chronic exercise training in AC5 KO mice (n=6) increased their performance further, as distance rose to 850±60m. However, a second month of exercise training did not increase WT performance significantly more. The next goal was to determine mechanisms. AC5 KO and trained WT mice shared similar gene expression in their gastrocnemius muscle with 183 genes commonly up‐ or down‐regulated. Shared gene ontology (GO) terms included important muscle regulatory gene sets including those involved in muscle contraction, metabolism, contractile fibers, and mitochondria. Both groups also had similar increases in oxidative fiber content in their gastrocnemius and extensor digitorum longus (EDL) muscle, indicating a fiber‐type switch in the exercising skeletal muscle. AC5 KO and trained WT mice both revealed increased SIRT1 protein levels in the gastrocnemius muscle and protection against oxidative stress. These results suggest that AC5 KO mice, without exercise training, perform better than even WT with exercise training and share similar mechanisms responsible for enhanced exercise capacity, and demonstrate considerably more reserve, allowing further improvement of performance with exercise training.
Aerobic Exercise Improves Nitric Oxide Bioavailability and Endothelium‐Dependent Vasorelaxation in Aortic Rings of Obese Zucker Rats
Authors: Lemaster Kent , DeVallance Evan , Branyan Kayla , Skinner Roy , Brooks Steve , Asano Shinichi , Pitzer Christopher , Olfert Mark , Bryner Randall , Frisbee Jefferson , Chantler Paul
Published: November 2023

Individuals with metabolic syndrome (MetS) have an increased aortic stiffness which is a strong, independent risk factor for cardiovascular disease. Additionally, MetS is associated with a decreased capacity for endothelium‐dependent, nitric oxide (NO)‐induced vasorelaxation which may contribute to the aortic stiffness and increased afterload on the heart. Obese Zucker rats, a translational rodent model of MetS, develop impaired vascular reactivity in aortic rings concomitant to the onset of MetS pathologies. Therapeutic interventions aimed at improving endothelial functioning are therefore considered important avenues for improving CV outcomes in MetS patients. We hypothesized that a chronic aerobic exercise intervention would improve endothelium‐dependent relaxation to methacholine in isolated aortic rings of obese Zucker rats. A total of 28 lean and obese Zucker rats (LZR and OZR, respectively) underwent 8 weeks of a treadmill exercise protocol beginning at 9 weeks of age. The animals were separated into 4 groups: sedentary LZR (LZR; n=7); sedentary OZR (OZR; n=6); exercise LZR (LZR‐EX; n=7); exercise OZR (OZR‐EX; n=7). Following the intervention, the thoracic aorta was dissected out from each animal and sectioned into rings, some of which were then mounted into an ex vivo wire tension myograph system. The remaining rings were evaluated for NO production in a DAF‐FM diacetate assay. Force transduction was used to measure the changes in aortic tension in response to pharmacological agonists. The aortic rings were mechanically set to 1 gram of tension then pre‐constricted using phenylephrine (1×10−6 μM), followed by a gradual dilation induced by increasing concentrations of methacholine (1×10−9, 1×10−8, 1×10−7, 1×106, 1×10−5 μM, respectively). Sedentary LZR demonstrated greater maximum methacholine‐induced vasorelaxation compared to sedentary OZR (83% vs. 67%) as well as reduced production of NO in the DAF assay. The exercise trained groups experienced significant improvements in maximal relaxation compared to their sedentary counterparts (LZR‐EX 93%; OZR‐EX 81%), as well as increased values of NO production measured in the DAF assay. These results demonstrate that 8 weeks of aerobic exercise enhances endothelial bioavailability and/or production of NO which improves endothelium‐dependent vasorelaxation in the aortas of both LZR and OZR. Exercise training may therefore be an important therapy for promoting greater arterial compliance in individuals with MetS.

Support or Funding Information

NIH

AHA
Ten Days of Free Wheel Running Alters Cortical Glutamate‐Linked Mitochondrial Respiration in Rat Brain
Authors: George Mitchell A. J. , Brebner Karen , Kane Daniel A.
Published: November 2023

The mechanisms through which exercise benefits brain function are not completely understood. Appreciation for the role of lactate as a major neuroenergetic fuel, particularly during exercise, has progressed in recent decades; however, the mitochondrial involvement in brain lactate metabolism is not clear. To explore the mechanisms involved, we examined mitochondrial lactate oxidation in brain and skeletal muscle from exercised rats. Respiratory oxygen flux (JO2) was measured in saponin‐permeabilized prefrontal cortex (PFC) and hippocampus (dentate gyrus; DG) samples, as well as red and white gastrocnemius muscle samples from five male Wistar rats (age: approximately 104 days) given access to running wheels. Over ten days of free wheel running, rats attained a maximal daily running distance averaging 2.1 ± 0.5 km. The wheel‐running rats remained relatively weight stable, losing 1.4 ± 3.1 g body mass on average, whereas the five paired control rats gained an average of 39.4 ± 2.8 g over the ten days. On day eleven, high resolution respirometry was performed on exercise and control samples ex vivo. The addition of lactate failed to affect mitochondrial JO2 (ADP + malate) from either brain or muscle in either group. However, JO2 rose significantly following the addition of exogenous NAD+ in each tissue (P < 0.01), implying the lactate dehydrogenase reaction operates outside of the mitochondrial matrix. This interpretation was further supported by inhibition of JO2 following addition of an inhibitor of pyruvate transport, UK‐5099 (P < 0.01). Interestingly, the subsequent addition of glutamate resulted in a disparate increase in mitochondrial JO2 among tissues and treatment. Compared to controls, glutamate‐supported mitochondrial JO2 in wheel runners was significantly greater in PFC (P < 0.05), but not DG. This rise in JO2 likely reflects activation of the malate‐aspartate shuttle through elevated mitochondrial glutamate transport. Paradoxically, wheel runners exhibited lower glutamate‐supported JO2 in red gastrocnemius (P < 0.05). This may be due to the influence of regular exercise on the putative lactate‐malate‐aspartate shuttle. The lactate‐malate‐aspartate shuttle is the interaction between the lactate and malate‐aspartate shuttles to translocate reducing power to the mitochondria. The decline in lactate dehydrogenase activity in skeletal muscle that accompanies aerobic exercise training is expected to negatively influence lactate‐malate‐aspartate shuttling, and may explain our observations in muscle. Collectively, the results of this study suggest that electron shuttling in rat brain following ten days of voluntary exercise is upregulated in PFC. These results also provide further evidence for an indirect mechanism of mitochondrial lactate oxidation in both brain and skeletal muscle.

Support or Funding Information

The Natural Sciences and Engineering Research Council of Canada; The Nova Scotia Health Research Foundation
Associations Between Physical Activity And Lower‐Limb Strength With Bone Parameters In Healthy Young Women: A Pilot Study
Authors: Murai Igor Hisashi , Dantas Wagner Silva , dos Santos Gil Saulo , Teixeira Eurimar Rogerio , Alvarenga Jackeline Couto , Benatti Fabiana , Pereira Rosa Maria Rodrigues , Roschel Hamilton , Gualano Bruno
Published: November 2023

Background

Mechanical loading‐induced strain provided from physical activity plays an essential role in promoting bone modeling. Furthermore, muscle contraction is an important factor in maintaining bone mass. Therefore, the study aimed to investigate the relationship between physical activity level and lower‐limb strength with bone mineral density (BMD), bone microarchitecture and body composition in young women.

Methods

Healthy young women (age: 29.0±6.2 yrs, BMI: 22.46±2.26 kg/m2, n=14), not engaged in systematic exercise training programs, participated in this cross‐sectional study. Moderate‐to‐vigorous physical activity (MVPA) was assessed by accelerometry (ActiGraph GT3X+) for seven consecutive days. Leg press one repetition maximum (1‐RM) test was conducted in order to evaluate lower‐limb strength. BMD (i.e. assessed in four different skeletal sites, as follows: whole body, total femur, femur neck and lumbar spine) and body composition (i.e. lean mass and bone mineral content [BMC]) were measured by dual‐energy x‐ray absorptiometry. Trabecular bone microarchitecture parameters were measured by high‐resolution peripheral quantitative computed tomography in left tibia.

Results

There was a positive correlation between MVPA and some skeletal sites, such as: total femur BMD (r=0.62, p=0.041) and femur neck BMD (r=0.63, p=0.038). No significant correlation was observed between MVPA and trabecular bone microarchitecture parameters. MVPA was significant correlated with lean mass (r=0.90, p<0.001) and BMC (r=0.78, p=0.004). Lower‐limb strength was positively correlated with whole body BMD (r=0.66, p=0.009), total femur BMD (r=0.76, p=0.001), femur neck BMD (r=0.69, p=0.006), lumbar spine BMD (r=0.74, p=0.002), as well as tibial trabecular number (Tb.N) (r=0.63, p=0.021), and a negative correlation between tibial trabecular separation (Tb.Sp) (r=−0.55, p=0.050). With regard to body composition, lower‐limb strength was significantly associated with lean mass (r=0.73, p=0.002) and BMC (r=0.83, p<0.001).

Conclusions

These preliminary data show that both lower‐limb strength and physical activity level are positively associated with measurements of BMD in young women, especially in lower‐limb sites. Furthermore, lower‐limb strength, but not physical activity level, is positively associated with tibial trabecular number and negatively associated with tibial trabecular separation.

Support or Funding Information

FAPESP (process: 2015/02833‐5)
Sciatic lesion does not induce bone disuse atrophy in a hibernating species
Authors: Utz Jenifer C. , Warsi Hina , Treat Michael , Sarkhanov Valeri , Thota Jagaeep , O'Toole Brendan , Donahue Seth , van Breukelen Frank
Published: November 2023

Bone loss is evident in as little as 2 weeks in common rodent models of disuse atrophy. Yet hibernating mammals are inactive for 6–9 months per year. We previously demonstrated the maintenance of bone strength following the hibernation season in ground squirrels. Here, we investigated if active season squirrels naturally resist bone disuse atrophy. Unilateral sciatic lesions were performed on squirrels collected from the wild in June and September. Following a 6‐week period of disuse, leg bones were collected and the following parameters were evaluated: bone morphometry, break force, flexural strength, flexural modulus, and ultrastructure (visualized through micro computed tomography). Despite sciatic denervation and disuse for 6 weeks, squirrels experienced no bone disuse atrophy. These results indicate resistance to bone disuse atrophy is not restricted to the hibernation season in ground squirrels.
A within‐subject comparison of changes in core temperature with hypoxia‐induced manipulations of relative exercise intensity
Authors: Coombs Geoff , Ravanelli Nicholas , Cramer Matthew , Imbeault Pascal , Jay Ollie
Published: November 2023

Traditionally, relative exercise intensity (i.e., %VO2max) has been thought to determine changes in core temperature during exercise in compensable environments; however, recent evidence from our laboratory has suggested otherwise. Using independent group designs, we have demonstrated that to make unbiased comparisons of changes in core temperature, exercise intensities should be set to elicit a fixed metabolic heat production (Hprod) per unit of total body mass (W/kg), irrespective of large differences in %VO2max (Jay et al. Am J Physiol. 2011) or absolute Hprod (Cramer & Jay, J Appl Physiol. 2014). Using a within‐subject design, the present study assessed whether changes in core temperature during exercise at a given Hprod in W/kg are altered by differences in %VO2max, secondary to acute hypoxia exposure (~13% O2) compared to normoxia (21% O2). It was also assessed whether the change in core temperature during exercise at a fixed %VO2max in hypoxic (HYP) conditions will be similar to normoxia (NORM) despite a 31±8% reduction in hypoxic VO2max and therefore a much lower Hprod. Four participants (1.78±0.04 m, 69.9±5.3 kg, 27±5 y) cycled for 45 min at 34°C, 45% RH in a climate‐controlled chamber. Measurements of esophageal temperature (Tes) and rectal temperature (Tre) were recorded every 5 s, and indirect calorimetry was recorded breath‐by‐breath. Preliminary data indicate that 45‐min ΔTes during exercise at a fixed Hprod (NORM: 6.3±0.4 W/kg; HYP: 6.9±0.4 W/kg; P=0.13) were similar between conditions (NORM: 0.52±0.22°C; HYP: 0.47±0.27°C; P=0.61), while ΔTre were also similar (NORM: 0.71±0.26°C; HYP: 0.74±0.27°C; P=0.41), despite large differences in relative exercise intensity (NORM: 40±8%VO2max; HYP: 62±8% VO2max; P=0.02). However, when a subsequent bout of hypoxic exercise was normalized for %VO2max (NORM: 40±8%VO2max; HYP: 46±4% VO2max; P=0.07), ΔTes in the hypoxic condition were lower compared to the normoxic condition (NORM: 0.52±0.22°C; HYP: 0.34±0.25°C; P<0.01) and ΔTre was lower as well (NORM: 0.71±0.26°C; HYP: 0.52±0.30°C; P=0.04) due to a lower Hprod (NORM: 6.3±0.4 W/kg; HYP: 5.3±0.6 W/kg; P=0.02). In conclusion, our within‐subject comparison provides further evidence that changes in core temperature are determined by exercise at a fixed Hprod in W/kg, despite large differences in relative exercise intensity, and that exercise at a fixed %VO2max, but different Hprod in W/kg, results in different changes in core temperature.

Support or Funding Information

This project was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada, and also in part by a research grant from the Gatorade Sports Science Institute.
TISSUE‐SPECIFIC AND CONCURRENT CIRCADIAN RHYTHM OF SIRT3 AND NICOTINAMIDE PHOSPHORIBOSYL TRANSFERASE EXPRESSION IN LIVER AND SKELETAL MUSCLE
Authors: Loehr Amanda R. , Dyer Normonique A. , Brandauer Josef
Published: November 2023

Nicotinamide phosphoribosyl transferase (Nampt) catalyzes the rate‐limiting reaction in nicotinamide dinucleotide (NAD) recycling. Nampt thus indirectly controls the activity of SIRT3, a mitochondrial NAD‐dependent protein deacetylase which regulates mitochondrial content and metabolism. Since Nampt is expressed in a circadian oscillation and many clock‐controlled acetylation modifications affect mitochondrial proteins, this suggests that SIRT3 is involved in circadian regulation of mitochondrial biology. We assessed the expression of Nampt and SIRT3 in various mouse tissues (liver, tibialis anterior, and diaphragm) at six time points of the 24‐hour cycle in an extended darkness (ED) period, i.e., in the absence of ambient light. 36 female ICR mice were maintained on a regular 12:12 hour light:dark cycle for 2 weeks. Following a 12‐hour dark period, mice were maintained in a dark environment for the remainder of the experimental period. Nampt and SIRT3 expression levels were positively correlated in all tissues (r, 0.526 to 0.797, all p < 0.05), but circadian variations were tissue‐specific. Liver Nampt and SIRT3 expression peaked 8‐hours into the ED period. Relative to peak expression, liver Nampt decreased by 24% at 12‐hours ED (p < 0.05) while SIRT3 expression decreased 26% at 16‐hours ED (p = 0.08). In tibialis anterior muscle, both Nampt and SIRT3 expression peaked at the beginning of the ED period. At 20‐hours ED, Nampt expression was 27% lower (p < 0.05) and and SIRT3 expression was decreased by 40% (p = 0.06). Conversely, both SIRT3 and Nampt expression in the diaphragm decreased over the first 8‐hours of the ED period but increased significantly by 20‐hours ED (Nampt, 50%, SIRT3, 72%, p < 0.01 for both). Our results suggest a tissue‐specific circadian regulation of mitochondrial proteins and energy metabolism that persists in the absence of regular light‐dark cycles.

Support or Funding Information

This work was supported by a grant to Gettysburg College from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program (AL), an Integrative Organismal Systems Fellowship by the American Physiological Society and a Kolbe Research Fellowship (ND), and a Gettysburg College Professional Development Grant (JB).
PGC‐1α4 Expression is Down‐regulated by ERK1/2 MAPK Signaling Axis of IL‐6
Authors: Brown Jacob , Rosa Megan , Lee David , Brown Lemuel , Perry Richard , Carson James , Dridi Sami , Washington Tyrone , Greene Nicholas
Published: November 2023

Loss of muscle mass and function are associated with reduced quality of life in many disease conditions. Recently, the peroxisome proliferator‐activated receptor γ coactivator‐1α4 (PGC‐1α4) isoform has been shown to promote gains in skeletal muscle mass; however the mechanism of regulation under normal and stress (inflammatory) conditions is still unknown.

PURPOSE

To elicit signaling mechanisms regulating the gene expression of Pgc‐1α4 during inflammation and particularly by IL‐6.

METHODS

Animal Experiments: Female C57BL/6J (WT) and C57BL/6J × IL‐6−/− (IL‐6 KO) mice were euthanized 10 weeks of age and tibialis anterior muscles were excised and snap frozen in liquid nitrogen. Tissue Culture: C2C12 myotubes were treated with recombinant IL‐6 (50 ng/ml), recombinant TNF‐α (20 ng/ml), Pyrrolidine dithiocarbamate (PDTC inhibitor of STAT 3 and NFκB, 50 μM), PD98059 (20 μM; MEK1/2 inhibitor preventing ERK1/2 activity), U0126 (10 μM; MEK1/2 inhibitor preventing ERK1/2 activity), and SB202190 (10 μM; inhibitor of p38 MAP kinase). RNA was isolated from WT and IL‐6 KO mice as well as C2C12 myotubes. RNA was analyzed by real time RT‐PCR for mRNA content analysis of 18S and Pgc‐1α4. Protein analysis was performed by immunoblot for P‐STAT3, STAT3, P‐ERK 1/2 and ERK 1/2 to confirm function of pharmacological inhibitors and normalized to Ponceau S. A student t‐test or one‐way ANOVA was employed as appropriate with α set at 0.05.

RESULTS

Pgc‐1α4 mRNA content was 10‐fold greater in IL‐6 KO mice compared to WT. In C2C12 myotubes, recombinant IL‐6 significantly repressed Pgc‐1α4 mRNA content by ~70% while Pgc‐1α4 content in TNF‐α and PDTC treated cells were not different from control. In the following experiments, 70% repression of Pgc‐1α4 mRNA by IL‐6 was reversed when IL‐6 was combined with ERK1/2 inhibition via MEK (PD098059 + IL6 and U0126 + IL‐6). To determine if MEK1/2 – ERK MAPK repression of Pgc‐1α4 mRNA is a specific function of ERK‐MAPK or a general function of MAPKs, the experiment was repeated with a p38 MAPK inhibitor (SB202190). As in the previous experiments, IL‐6 suppressed Pgc‐1α4 mRNA by ~70%; moreover, p38 MAPK inhibition by SB202190 did not affect the IL‐6 repression of Pgc‐1α4 mRNA. Confirming the pharmacologic function, a significant 40% increase in the P‐STAT3/STAT3 ratio and the P‐ERK/ ERK ratio with IL‐6 treatment was observed, while inhibitors of STAT3 (PDTC) and ERK 1/2 (PD098059) significantly decreased these ratios by 30% and 50 % when compared to vehicle, respectively.

CONCLUSION

PGC‐1α4 expression is repressed by IL‐6 through the ERK1/2 signaling axis. These findings may have significant impact in treatment to prevent and ameliorate muscle wasting conditions.

Support or Funding Information

Funded by the University of Arkansas
Dexamethasone Promotes the Long‐Term Functional Recovery of Neuromuscular Junction after Ischemia/Reperfusion Injury Induced by Tourniquet in Mouse Hindlimb
Authors: Zhang Dongze , Wang Dong , Muelleman Robert L. , Li Yu‐Long
Published: November 2023

Tourniquet application is an effective means of arresting life‐threatening limb hemorrhage in pre‐hospital setting. However, tourniquet‐induced ischemia and subsequent reperfusion cause serious ischemia‐reperfusion (IR) injury for the neuromuscular junction and limb. Although accumulating evidence has uncovered protective effects of anti‐inflammation and anti‐oxidation on acute IR injury in skeletal muscles, it is unclear whether these therapies improve long‐term functions of the neuromuscular junction and skeletal muscle. Our current study investigated if Dexamethasone (Dex) promotes repair of the neuromuscular junction and restore the skeletal muscle contractile function in tourniquet‐induced hindlimb IR. Unilateral hindlimb of C57/BL6 mice was subjected to 3‐hours of ischemia following 6‐weeks of reperfusion (6‐wk‐IR) via placement and release of a tourniquet at the hip joint. To test the protective effect of Dex, Dex treatment (1mg/kg/day, i.p.) began on the day of IR induction and lasted for varied periods including 1 day, 3 days, 1 week, 2 weeks, 4 weeks and 6 weeks. On the day of the terminal experiments, gastrocnemius muscle contraction was detected under sciatic nerve stimulation (10 V, 50 Hz, 5 sec) in situ. Function of the neuromuscular junction was measured using electrophysiological recording of endplate potentials (EPP) in situ. Western blot was used to detect the protein expression of nAchR α1 and nAchR β1 in gastrocnemius muscles. The amplitude of EPP was lower in mice with 6‐wk‐IR than that in sham mice. Additionally, sciatic nerve‐stimulated gastrocnemius muscle contraction in mice with 6‐wk‐IR was 60% of normal skeletal muscle contraction recorded in age‐matched sham mice. Dex treatment for 1 or 3 days did not significantly increase amplitude of EPP and improve sciatic nerve‐stimulated gastrocnemius muscle contraction in tourniquet‐induced 6‐wk‐IR. Dex treatment for 1‐week exerted a maximum effect on increasing amplitude of EPP and improving skeletal muscle contraction, which were gradually becoming less, and even deleterious, with prolonged Dex treatment. Furthermore, there are no significant differences in protein expression of nAchR α1 and nAchR β1 in gastrocnemius muscle between groups. These results suggest that 1‐week is an optimum duration for application of Dex to promote repair of neuromuscular junction and improve long‐term recovery of IR‐induced skeletal muscle contraction. Simultaneously, these results also indicate the toxic effect of Dex on the neuromuscular junction and skeletal muscle after prolonged treatment of Dex.
Changes in Titin Isoform Composition Following Knockout of Bmal1, a Core Molecular Clock Factor, in Skeletal Muscle
Authors: Riley Lance A , Zhang Xiping , Esser Karyn A
Published: November 2023

Recent work from our lab has shown that disruption of the endogenous molecular clock mechanism in skeletal muscle is sufficient to induce muscle weakness with changes in fiber type and fibrosis. To begin to discern the molecular mechanisms that link the changes in the molecular clock with changes in muscle function, we tested whether the muscle of inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1−/−) mice would also exhibit changes in sarcomeric protein expression. One of the proteins we focused on was titin, the giant filamentous protein that maintains sarcomeric structure, underlies passive tension, and serves as a scaffold for many proteins and signaling molecules. In our studies, we determined that the tibialis anterior muscle of the iMSBmal1−/− mice exhibits a significant increase in the longer, more compliant isoform of titin (38% long isoform/total titin in iMSBmal1−/− mice vs. 19% in iMSBmal1+/+ mice). While there is no change in the total amount of titin expressed, comparisons of results from a microarray data set between the two treatment groups indicate that probe sets covering the PEVK region were significantly up‐regulated in the gastrocnemius muscle of these mice. This region of titin pre‐mRNA is known to be spliced out in the presence of the RNA binding protein RBM20. Using this logic, we noted that expression of Rbm20 mRNA is decreased approximately 24% in iMSBmal1−/− gastrocnemius muscle compared to iMSBmal1+/+ controls suggesting that the molecular clock could be acting through this protein to modify titin alternative splicing. Ongoing studies are focusing on the connection between the molecular clock and RBM20 and if the change in RBM20 expression is responsible for the difference in titin spliceform expression.

Support or Funding Information

NIH AR066082
Measures of Fiber‐type Specific Transcriptional Regulation of Mitochondrial Morphology
Authors: Hambleton Catherine A , Trevino Michael , Herda Trent , Gallagher Philip M.
Published: November 2023

Mitochondrial homeostasis includes turnover of fusion and fission proteins. Silent information regulator (SIRT) 1, peroxisome‐proliferator‐activated receptor‐gamma co‐activator‐1 (PPARγ), and peroxisome proliferator‐activated receptor (PPAR)‐γ‐coactivator 1 alpha (PGC‐1α) are suggested to be transcriptional master co‐regulators of mitochondrial biogenesis, metabolism, and key factors in suppression of oxidative stress. Higher expression of PGC‐1α and SIRT1 are found in type‐I oxidative fibers and have been linked to fiber‐type shifting fast‐to‐slow twitch in skeletal muscle. The purpose of this study was to determine differential gene expression of mitochondrial turnover in type‐II and type‐I single muscle fibers. Eleven subjects volunteered for a biopsy obtained from the vastus lateralis muscle. Fiber‐type specific samples were used to quantify the gene expression from individual fibers. Myosin heavy chain (MHC) analysis was performed using single muscle fibers isolated from the muscle bundle. From each individual fiber, one‐third each was placed in buffer for fiber typing and the other two‐thirds of each fiber was placed into reverse transcriptase (RT) buffer to determine the MHC isoform. Once the fiber types were identified, cDNA from each fiber type was combined for a given subject. After the reverse transcriptase reaction, the cDNA for each individual fiber was used for each of the qPCR reactions to determine expression levels. Data was analyzed using a paired sample t‐test (p<0.05). ΔΔCT analysis revealed no significant differences in SIRT1, PGC‐1α, PPARγ, OPA1 and PINK‐1 expression between type‐II and type‐I fibers. In conclusion, this study found no difference in resting levels of mitochondrial turnover in type‐II and type‐I single muscle fibers.
Combined Oxandrolone and Propranolol Therapy Attenuate Protein Breakdown in Severely Burned Pediatric Patients
Authors: Chao Tony , Herndon David N , Porter Craig , Malagaris Ioannis , Abdelrahman Doaa Reda , Suman Oscar E , Sidossis Labros S
Published: November 2023

Introduction

Severe burn result in prolonged hypermetabolism and increased skeletal muscle catabolism. Propranolol has been shown to blunt the hypermetabolic stress response to burns while oxandrolone was shown to improve muscle protein synthesis efficiency. However, the effect of combined oxandrolone and propranolol therapy on skeletal muscle fractional synthesis (FSR) and breakdown (FBR) rates are not known. The objective of this study is to determine the effects of oxandrolone and propranolol (oxprop) treatment on skeletal muscle protein turnover in severely burned pediatric patients. In this placebo controlled clinical trial, we hypothesize that oxprop will improve skeletal muscle protein turnover in pediatric burn patients.

Method

We studied 24 pediatric patients with severe burns (≥ 30% total body surface area; TBSA) during their inpatient hospitalization. Patients were randomized to placebo (14 control) or oxprop (10). Protein kinetics were determined with bolus injections of 13C6 and 15N phenylalanine at 2 and 4 weeks post‐injury. Skeletal muscle biopsies were obtained from the m. vastus lateralis. Plasma and muscle intracellular and bound protein pool enrichments were determined by gas chromatography‐mass spectrometry (GCMS). A Mann‐Whitney test was performed between groups. Significance was determined at p<0.05.

Results

Patients were similar in age (8 ± 6yr) and TBSA (53 ± 17% vs 48 ± 8%) between the control and oxprop groups. Muscle FSR was not different between control and oxprop at 2 weeks (0.13 ± 0.04%/h vs 0.08 ± 0.01%/h, p=0.73) or 4 weeks (0.16 ± 0.03%/h vs 0.13 ± 0.02%/h, p=0.92). FBR was not different between control and oxprop at 2 weeks (0.27 ± 0.05%/h vs 0.20 ± 0.02%/h, p=0.27). At 4 weeks, FBR in the oxprop group was significantly less than control (0.14 ± 0.02%/h vs 0.28 ± 0.04%/h, p<0.05). Protein net balance was not different between control and oxprop at 2 weeks (−0.14 ± 0.03%/h vs −0.12 ± 0.03%/h, p=0.33). At 4 weeks, net balance was improved in oxprop versus control (−0.01 ± 0.02%/h vs −0.12 ± 0.04%/h, p<0.05).

Conclusion

Combined oxandrolone and propranolol therapy significantly improves skeletal muscle protein turnover in pediatric burn patients after 4 weeks of treatment. The improvement in skeletal muscle protein net balance with oxprop treatment is achieved by blunting protein breakdown. Treatment with oxandrolone and propranolol for 4 weeks can blunt the catabolic effect seen in severe burn injury.

Support or Funding Information

NIH (P50 GM060338, R01 GM056687, R01 HD049471 and T32 GM008256), NIDILRR (90DP00430100) and Shriners Hospitals for Children (84080, 84090, 71006, 71008 and 71009) grants.
The Impact of Acetaminophen Consumption Prior to Resistance Exercise on the Initial Myogenic Gene Response in Human Skeletal Muscle
Authors: D'Lugos Andrew , Patel Shivam , Eldon Erica , Carroll Chad , Dickinson Jared
Published: November 2023

Resistance exercise is a powerful stimulus for skeletal muscle adaptation. Previous data have shown that cyclooxygenase (COX) inhibiting drugs may interact with the cellular mechanisms regulating the adaptive response of skeletal muscle. The purpose of this study was to determine if prior consumption of the COX‐inhibitor acetaminophen (APAP) alters the immediate myogenic gene response in skeletal muscle following resistance exercise (RE). In a double‐blinded, crossover design, healthy young men (n=4) performed two trials of single‐legged RE (8 sets, 10 reps, 70% max strength). Subjects ingested either APAP (1000mg/6h) or placebo (PLA) for 24h prior to RE (final dose consumed immediately post RE). RT‐qPCR was used to examine mRNA expression of myogenic precursors in skeletal muscle (vastus lateralis) biopsies obtained at rest and at 1, and 3h post RE. Relative to basal values, DLK1 mRNA expression was increased at 1h post RE in both groups (P<0.05). VEGFA mRNA expression was increased 1h post RE in both groups, and remained elevated 3h post RE only in APAP (P<0.05). MYOG mRNA expression was increased at 3h post RE only in PLA (P<0.05), whereas MGF mRNA expression was decreased 3h post RE only with APAP (P<0.05). MYF5, MYF6, and PAX7 mRNA expression were unchanged in both groups and at all time points (P>0.05). These preliminary data indicate that APAP consumption prior to RE may induce subtle alterations in the immediate myogenic gene response to heavy resistance exercise. These data further highlight a unique interaction between the consumption of COX‐inhibiting drugs and the processes that regulate skeletal muscle adaptations to exercise.