bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Tripartite Motif E3 Ubiquitin Ligases Facilitate Skeletal Muscle Atrophy
Authors: Manring Heather , Smith Evan M. , Beck Eric X , Mcelhanon Kevin E. , Gushchina Liubov , He Wei A. , Guttridge Denis C. , Weisleder Noah
Published: November 2023

Muscle atrophy involves a loss of muscle mass and decreased contractile force production as part of the pathophysiology of many conditions including heart failure‐ or cancer‐induced cachexia, disuse atrophy, and age‐related sarcopenia. Decreased muscle fiber size and other mechanisms contribute to a loss of muscle strength resulting in frailty that compromises the health and well‐being of patients. During heart failure the degree of cachexia more closely correlates with fatigue level than the ejection fraction of the heart, and over 20% of cancer patients suffer diminished quality of life and eventually succumb to cachexia symptoms rather than as a direct result of the cancer itself. Atrophy requires ubiquitination of many muscle proteins through the action of E3 ubiquitin ligases. Two key E3 ligases, MURF1 and MAFbx/Atrogin‐1, are upregulated in several types of muscle atrophy and are essential for the atrophic response to occur during a variety of different stimulus. MURF1, also known as TRIM63, belongs to the large family of tripartite motif (TRIM) E3 ubiquitin ligases suggesting that other TRIM family members may have important roles during atrophy. Since many proteins in the muscle fiber must be ubiquitinated through targeting by specific E3 ligases, multiple novel E3 ligases must be required for muscle atrophy to occur. There are several candidate TRIM proteins that could potentially contribute to muscle atrophy. TRIM72/MG53 is on potential candidate E3 ligase as it is implicated in myoblast differentiation and muscle hypertrophy while its loss leads to susceptibility to cell membrane injury. However, little is known regarding the function of TRIM72 in muscle atrophy. Our current study investigates the role of TRIM72 in multiple models of muscle atrophy. Mouse models of cancer‐induced cachexia, dexamethasone induced muscle atrophy, and hind limb unloading were utilized in these experiments. Our findings show increased expression of TRIM72 protein in tibialis anterior and soleus muscles isolated from these mouse models of muscle atrophy. To evaluate the functional role of TRIM72 in skeletal muscle atrophy, muscles from trim72−/− and wild type mice following 12 days of hindlimb unloading were evaluated. Muscles from trim72−/−mice are resistant to disuse atrophy as these mice lost a lower percentage of muscle mass than wild type mice. Similarly, studies using dexamethasone to induce atrophy indicate decreased atrophy in trim72−/− muscles compared to wild type mice. These results establish that TRIM72 expression increases in parallel to other atrophy‐linked E3 ligases and that the expression of TRIM72 is necessary for the efficient atrophy of skeletal muscle following various muscle wasting stimuli. Targeting TRIM72 and other related E3 ubiquitin ligases could potentially act as a therapeutic approach for the treatment of different forms of muscle atrophy.
Effects of PGC‐1α Overexpression on Glucocorticoid‐induced Atrophy Signaling in C2C12 Muscle Cells
Authors: Rahnert Jill A , Perry Ben D , Zheng Bin D , Price Russ
Published: November 2023

Muscle wasting (atrophy) is a debilitating consequence of many catabolic conditions and has been linked to decreased expression of the transcriptional coactivator PGC‐1α. In some models of atrophy, overexpression of PGC‐1α attenuates muscle loss, in part, by reducing FoxO‐mediated atrophy gene expression. FoxOs are transcription factors regulated primarily by phosphorylation via Akt. Previous studies indicate that activation of Akt prevents downstream FoxO signaling during atrophy and that overexpression of PGC‐1α increases phosphorylation (i.e. inhibition) of FoxO proteins. The goal of this study was to determine whether increasing PGC‐1α protein preserves Akt phosphorylation using two in vitro models of atrophy, starvation and glucocorticoid treatment. C2C12 myotubes were infected with an adenovirus encoding GFP (control) or PGC‐1α followed by incubation in control differentiation media (DMEM + 2% horse serum), serum‐free DMEM (SF) to induce starvation, or differentiation media with the synthetic glucocorticoid, dexamethasone (Dex, 1uM), for 24h. Infection of cells with the PGC‐1α adenovirus increased PGC‐1α mRNA and protein and augmented Akt phosphorylation. In GFP‐expressing myotubes, SF reduced PGC‐1α protein and Akt phosphorylation and overexpression of PGC‐1α attenuated the starvation response. Dex did not alter PGC‐1α protein or Akt phosphorylation, however, it increased the FoxO target gene, Atrogin‐1. In addition, overexpression of PGC‐1α increased PGC‐1α protein in Dex‐treated cells and attenuated the increase in Atrogin‐1. These data suggest that maintenance of Akt phosphorylation may contribute to the protective effect of PGC‐1α. Furthermore, they underscore the role of PGC‐1α in the preservation of muscle protein during glucocorticoid‐mediated atrophy.

Support or Funding Information

Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit I01BX001456
Comparing Atrophy‐Inducing Signals on ER Stress in C1 C1 2 Muscle Cells
Authors: Newberry Erin R , Rahnert Jill A. , Price Russ
Published: November 2023

Muscle wasting (atrophy) is a consequence of chronic diseases, such as kidney disease or diabetes, and reduces the patient's overall quality of life and functional independence. Previous studies in this lab show that muscle cells treated with palmitate (Palm, a saturated fatty acid) activates ER stress and contributes to the atrophy process. The purpose of our experiments is to determine whether ER stress is a common feature of atrophy signaling by comparing the effects of two known inducers of muscle atrophy, glucocorticoids and Palm, on ER stress markers. Glucocorticoids are elevated during physiological stress like chronic diseases and are permissive for muscle wasting. Therefore, we tested the hypothesis that glucocorticoids increase markers of ER stress. C1 C1 2 muscle cells were treated with 1μM Dexamethasone (DEX, a glucocorticoid) and/or 500μM of Palm. The mRNA levels of the ER stress markers ATF4 and CHOP were measured by qPCR methods. ATF4 represents one of three arms of the ER stress response and CHOP represents an integrated response of all three arms. Treating myotubes with DEX had little effect on ATF4 or CHOP mRNA levels compared to the control. Palm did not change ATF4 mRNA but, similar to previous work, Palm increased CHOP mRNA. Co‐treatment of Palm and Dex synergistically increased both ATF4 and CHOP. These results suggest that in the presence of other atrophy signals, glucocorticoids act synergistically to enhance ER stress signaling. Future studies will evaluate whether activation of ER stress in co‐treated cells prolongs cell survival.

Support or Funding Information

R25DK101390, VA‐MERIT I01BX001456, NIH R01 DK 95610, NIH T32 DK 007656
IL‐33 Receptor Gene Deletion and Skeletal Muscle Abnormalities in a Mouse Model of Pulmonary Arterial Hypertension
Authors: Cannon Daniel T , Gutierrez Alma , Tsui Tzuhan , Lerner Andrew D , Breen Ellen C , Bigby Timothy D
Published: November 2023

Pulmonary arterial hypertension (PAH) is a progressive, incurable disease of the pulmonary vasculature, leading to high pulmonary vascular resistance and right ventricular failure. In addition, PAH patients show marked skeletal muscle abnormalities that may develop before right heart pathology. Thus, skeletal muscle dysfunction may be more closely related to the primary pathology of PAH than originally thought. We hypothesized that IL‐33, recently implicated in the cardiopulmonary pathogenesis of PAH, is also requisite for skeletal muscle maladaptation.

Using adult C57BL/6J mice, we induced PAH with 3 wks of chronic hypoxia (FiO2 0.10) and weekly subcutaneous injections (20 mg.kg−1) of the VEGF receptor kinase inhibitor Su5416 (SuHx). SuHx was applied in both wild‐type (WT) and IL‐33 receptor gene ablated (ST2−/−) mice. Further, vehicle control (DMSO) WT mice were generated using the same injection volume and schedule and were housed in normoxia (CON). Mitochondrial function was measured in permeabilized diaphragm and soleus using a titration protocol that included maximal ADP‐stimulated respiration (JO2max) and non‐phosphorylating electron transport system capacity (ETS).

Diaphragm JO2max was reduced in WT‐SuHx vs. WT‐CON (85±26 vs 162±62 pmol.s.mg‐−1; p<0.05), but not in ST2−/−‐SuHx (148±102 pmol.s.mg‐−1; p>0.72 vs WT‐CON). Diaphragm ETS followed a similar pattern in WT‐SuHx vs WT‐CON (90±29 vs 167±63 pmol.s.mg‐−1; p<0.05), and was not different in ST2−/−‐SuHx vs WT‐CON (152±104 pmol.s.mg‐−1; p>0.73). Flux control ratios (JO2max/ETS) were not different between groups (p>0.75). Soleus JO2max was reduced in both WT‐SuHx and ST2−/−‐SuHx vs WT‐CON (70±33 and 86±38 vs 140±40 pmol.s.mg‐−1; p<0.05). Soleus ETS was also reduced in WT‐SuHx and ST2−/−‐SuHx vs WT‐CON (68±24 and 86±38 vs 140±30 pmol.s.mg‐−1; p<0.05). Similar to diaphragm, soleus flux control ratios were not different between groups (p>0.73).

The SuHx model showed a profound reduction in skeletal muscle mitochondrial oxidative capacity, suggestive of similar abnormalities to those reported in patients with PAH. As the flux control ratios were not different in SuHx, reduced mitochondrial volume‐density was a more likely mechanism, rather than a deficit in the quality of integrated function between electron transport and phosphorylation. Global gene deletion of the IL‐33 receptor (ST2) prevented diaphragm mitochondrial deficits in SuHx, but soleus muscle was less well‐protected. Our data strengthen the hypothesis that inflammatory cytokines, such as IL‐33, may play a critical role in skeletal muscle maladaptation to PAH.

Support or Funding Information

This research was supported by the US Department of Veterans Affairs.
Chronic Heat Preconditioning Enhances Skeletal Muscle Function during Hypoxia and Reoxygenation
Authors: Zuo Li , Roberts William J. , Pannell Benjamin K.
Published: November 2023

Skeletal muscle that is exposed to hypoxia followed by reoxygenation (H/R) is susceptible to dysfunction and fatigue. However, the mechanisms that underlie skeletal muscle fatigue during H/R are not completely understood. The purpose of our study was to determine the role that chronic heat preconditioning (CHP) may play in protecting skeletal muscle against the deleterious effects of H/R. We hypothesized that CHP would mitigate skeletal muscle fatigue and attenuate ROS formation during H/R. During CHP treatment, C57BL/6 mice were housed in a heating chamber at 42°C for 30 minutes. Next, the mice were transferred to another chamber at room temperature for 30 minutes. This cycle was repeated 3 times per day, 5 days/week, for 2 weeks. Following CHP, mice were sacrificed and their diaphragms were removed for muscle function analysis. The muscle strips were electrically stimulated in a contraction chamber filled with Ringer's solution during H/R. Our data revealed that CHP significantly increased muscle function during hypoxia (31.5 ± 1.7% for treated vs. 13.1 ± 2.7% for control, p < 0.05; expressed as percent of the maximal force at the end of the contraction protocol) and also showed a trend to increase muscle fatigue resistance during reoxygenation. We monitored cellular levels of reactive oxygen species (ROS) via confocal microscopy by utilizing dihydrofluorescein diacetate (Hfluor‐DA), which is a fluorescent probe sensitive to intracellular ROS. Compared to the non‐treated group, CHP‐treated muscle strips demonstrated mitigated ROS fluorescence at the end of both hypoxia and reoxygenation periods, respectively (p < 0.05). Our results suggest that CHP may play a protective role against muscle fatigue and ROS formation during H/R in skeletal muscle.

Support or Funding Information

Supported by OU General Fund G110
Electromechanical Delay and Peripheral Fatigue: Partitioning the Contributors during Electrically Evoked Contractions
Authors: Esposito Fabio , Monti Elena , Rampichini Susanna , Limonta Eloisa , Venturelli Massimo , Cè Emiliano
Published: November 2023

Skeletal muscle contraction is a phenomenon involving several mechanisms that are electrochemical and mechanical in nature. The time lag between muscle electrical activation and the beginning of force production has been previously defined as the electromechanical delay. A novel electromyographic (EMG), mechanomyographic (MMG) and force (F) combined approach can provide the duration of the electrochemical and mechanical components of the total delay (DelayTOT). Prior investigations evaluated the acute effects of a fatiguing protocol on DelayTOT. The aim of this study was to evaluate the time course of the changes in the different DelayTOT components during a fatiguing stimulation. To this purpose, EMG, MMG and F signals were detected on the medial gastrocnemius muscle in eighteen male participants (age: 25±3 yrs; body mass: 77±4 kg; stature: 1.81±0.06 m; mean±SD) during a fatiguing protocol (twelve continuous stimulations at 35 Hz, 9.5 s on/0.5 s off; total time: 120 s) under isometric condition. Stimulation current (Stim) and EMG, MMG and F signals were recorded during contraction. The total electromechanical delay during contraction (DelayTOT) and its three components (between Stim and EMG: Δt Stim‐EMG, synaptic component; between EMG and MMG: Δt EMG‐MMG, electrochemical component; and between MMG and F: Δt MMG‐F, mechanical component) were calculated. Before fatigue DelayTOT was 22.3±0.9 ms. Δt Stim‐EMG, Δt EMG‐MMG and Δt MMG‐F relative contribution to DelayTOT was 6%, 48% and 46%, respectively. During fatigue, DelayTOT and Δt MMG‐F lengthened significantly in the first 40 s, then remaining stable, whereas Δt Stim‐EMG and Δt EMG‐MMG increased until the 10th and 30th s, respectively. At the end of the fatiguing protocol, F decreased by 44%. DelayTOT was 31.2±1.6 ms. Δt Stim‐EMG, Δt EMG‐MMG and Δt MMG‐F significantly changed their contribution to 5%, 43% and 53%, respectively. During a fatiguing stimulation, the present EMG, MMG and F combined approach unveiled a different effect of peripheral fatigue with time on the synaptic, electrochemical and mechanical components.

Support or Funding Information

The study has been funded by the Department of Biomedical Sciences for Health, University of Milan
The Molecular‐Clock and MyoD1 Work in a Synergistic Fashion to Promote Robust Circadian Expression of Titin‐cap in Skeletal Muscle
Authors: Hodge Brian Andrew , Zhang Xiping , Arnold Nick , Esser Karyn A
Published: November 2023

Pressures caused by daily oscillations in the environment have favored the evolution of organisms that have adopted 24‐hour rhythmic cycles, or circadian rhythms. Daily oscillations in mammalian physiology are generated through an intrinsic molecular‐clock mechanism. An unresolved phenomenon in the field of circadian rhythms is the underlying mechanism(s) that allow the core molecular‐clock proteins (Bmal1/Clock common across all cell types) to transcriptionally target genes in a tissue‐specific fashion. The goal of this project was to use a targeted approach in skeletal muscle to identify mechanisms in which the molecular‐clock regulates the transcription of skeletal muscle specific genes. We focused our study on the molecular regulation of the muscle specific z‐line gene Titin‐cap (T‐cap). We choose this gene because it is highly circadian in skeletal muscle, it is known to be regulated downstream of the myogenic regulatory factor MyoD1 in skeletal muscle and its expression is significantly down‐regulated in the muscle of Bmal1 KO mice. Utilizing T‐cap promoter‐reporter contstructs in C2C12 myotubes, we demonstrate that the T‐cap promoter contains essential elements required for circadian oscillation in vitro (absence of environmental time cues) suggestive of a direct clock‐controlled gene. We performed a series of transfection experiments testing T‐cap promoter‐reporter constructs in C2C12 myotubes, and demonstrated that T‐cap is transactivated by overexpression of clock factors BMAL1:CLOCK, as well as MyoD1. We demonstrate that the T‐cap promoter contains essential elements required for circadian oscillation in vitro (absence of environmental time cues) suggestive of a direct clock‐controlled gene. Overexpression of MyoD1 was sufficient to enhance the amplitude of the T‐cap promoter‐reporter rhythm without affecting the circadian phase or period length. Interestingly, co‐transfection studies with mutant forms of Bmal1 or MyoD1, showed that BMAL1:CLOCK and MyoD1 work in a synergistic fashion and require each other to promote the circadian expression of T‐cap in C2C12 myotubes. Deletion constructs and mutagenesis studies followed by ChIP assays demonstrated that both BMAL1 and MyoD1 were bound to the proximal promoter of T‐cap, and we identified a tandem E‐box element required for T‐cap circadian expression. These results demonstrate that the molecular‐clock factors, BMAL1 and CLOCK, work with MyoD1, a tissue‐specific transcription factor, to promote the circadian expression of T‐cap in skeletal muscle. On going studies are aimed at elucidating the molecular basis underlying the synergist interaction observed between the molecular‐clock and MyoD1.

Support or Funding Information

NIH RO1: AR066082
The Muscle Force Characteristics of SEMG in The Take‐off Phase of 100 m Hurdles Female Athlete
Authors: Sun Youping , He Jing , Wang Li
Published: November 2023

OBJECTIVE

Reveal the characteristics of muscle force of 100 m hurdles female athlete in

METHODS

Surface electromyography (sEMG) signals are widely used to evaluate muscle force, In this work, a100 m hurdles female athlete has been made to analyze the complexity of sEMG signals in take‐off phase. 16 muscle were Selected in study, for example the deltoid toe, beam under the rectus, vastus lateral's muscle, erector spinae, gluteus maximums muscle, semitendinosus muscle, medial head of gastrocnemius muscle, tensor fascia lata and so on.

RESULTS

The sequence major muscle forcibly of the hurdle step skill in different stages as follows. In take‐off phase: the under rectus abdomen of the lead leg ←the glute maximus of the swinging leg ←the semitendinosus muscle of the lead leg← the deltoid toe of the lead leg. In flight phase: the under rectus abdomen of the lead leg ←the tensor fasciae latae muscle of the swinging leg ←the glute maximus of the swinging leg←the deltoid toe of the lead leg. In touch‐down phase: the glute maximus of the swinging leg ←the musculus semitendinosus of the swinging leg←the musculus semitendinosus of lead leg←the deltoid toe of the swinging leg.

CONCLUSION

It is found that the under rectus abdomen of the lead leg is the major muscle in take‐off phase, and the under rectus abdomen of the lead leg is the major muscle in flight phase, and the glute maximus of the swinging leg is the major muscle.

Support or Funding Information

The key lab of adolescent health intervention and exercise assessment, Ministry of China
Chronic Binge Alcohol (CBA) Administration Increases Protein Tyrosine Phosphatase 1B (PTP1B) Protein Expression in Fed Simian Immunodeficiency Virus (SIV) Infected Rhesus Macaques
Authors: Ford Stephen M. , Simon Liz , Stouwe Curtis Vande , Allerton Tim , Dufour Jason P. , Nelson Steve , Bagby Gregory J. , Molina Patricia E.
Published: November 2023

Chronic alcohol consumption, HIV infection, and anti‐retroviral therapy (ART) have independently been shown to impair whole body insulin responsiveness. Previous studies from our group have shown that skeletal muscle of CBA administered SIV‐infected (CBA/SIV) male rhesus macaques has upregulated ubiquitin‐proteasome pathway activity, enhanced pro‐inflammatory cytokine, and suppressed IGF‐I expression at end stage SIV infection. These findings suggest that CBA may impair insulin‐mediated anabolic responses at end‐stage disease. Whether systemic and skeletal muscle metabolic alterations are present at earlier time points of SIV infection or are affected by ART administration was not known. In this study, we tested the hypothesis that CBA decreases whole body insulin sensitivity and insulin signaling in the skeletal muscle during the asymptomatic stage of SIV infection in male rhesus macaques and proposed that this would be further aggravated by ART. CBA or sucrose (SUC) was administered intragastrically for 3 mos prior to inoculation with SIVmac251 and continued until necropsy. At 2.5 mos. post‐SIV infection, daily administration of ART (tenofovir and emtricitabine) was initiated in half of the SUC/SIV and CBA/SIV macaques. Frequently sampled intravenous glucose tolerance tests were performed to analyze glucose‐insulin dynamics, including acute insulin response to glucose (AIRg), disposition index (DI), and insulin sensitivity index, using a homeostatic model. One week prior to necropsy, a controlled feeding protocol was performed with infusion of a Carnation breakfast supplement (9.283 mL/kg/h) beginning 1.5 hours after CBA or SUC administration, continued for one hour, followed by a quadriceps femoris biopsy. One week after the feeding protocol, fasted‐state muscle biopsy was collected. At study endpoint (14 mo CBA, 11 mo SIV) CBA/SIV macaques had a significantly reduced (p<0.05) AIRg and DI and circulating levels of adiponectin, relative to SUC/SIV macaques, irrespective of ART status. Western blot analysis of fasted‐and fed‐state skeletal muscle samples revealed a statistically significant increase in fed‐state PTP1B, a negative regulator of insulin signaling, in CBA/SIV relative to SUC/SIV macaques and irrespective of ART status. These findings suggest that CBA impairs insulin‐mediated effects in CBA/SIV macaques and warrant future studies elucidating the specific alterations in the insulin signaling cascade that are modulated by CBA.

Support or Funding Information

The work was supported by National Institutes of Health (NIH) grants: P60AA09803, T32AA07577, and F30AA024030‐01A1.
Quantification and Characterization of Type I Myofiber Grouping: the Effect of Aging
Authors: Hammond Kelley G , Kelly Neil A , Bickel C. Scott , Bamman Marcas M
Published: November 2023

Among the neuromuscular changes that occur with aging, older adults may experience neuronal death, which is revealed through sarcopenia and motor unit loss. However, motor unit loss may not always elicit myofiber death, as some denervated myofibers are reinnervated by another local motor neuron and incorporated into that motor unit. This reorganization of motor units can facilitate the integration of type IIa myofibers into a type I motor unit, causing those fibers to express slow myosin heavy chain (MHC; type I). This denervation/reinnervation and subsequent myofiber type shift can result in type I myofiber grouping.

PURPOSE

To characterize grouped versus ungrouped type I myofibers by morphological and functional features in older adults.

METHODS

Muscle tissue specimens were collected from the vastus lateralis, and myofibers were assessed by MHC expression (I, IIa, IIx) for type, size, and grouping. Grouping was determined using a statistical model which includes type I distribution and nearest‐neighbor methods. Capillarization was assessed by immunohistochemistry.

RESULTS

Percent of grouped type I myofibers is higher in Old compared to Young (51.7% vs. 21.4%; p<0.001). The average number of myofibers per group is higher in Old than Young adults (56.6 vs 13.3; p=0.01). In Older adults, grouped type I myofibers are significantly larger than ungrouped (4911 vs. 4335 μm2; p<0.001), and have fewer capillary contacts per myofiber (2.4 vs. 3.7; p<0.001).

CONCLUSION

Age‐related changes to skeletal muscle includes an increase in type I myofiber grouping. This grouping is characterized by increased myofiber size and decreased capillarity in grouped type I myofibers.

Support or Funding Information

Supported by NIH Grant T32HD071866 and the UAB Center for Exercise Medicine
Does Titin Explain The High Passive Force Observed in Frog Tibialis Anterior Muscle?
Authors: Joumaa Venus , Kim Si Yong , Seerattan Ruth , Herzog Walter
Published: November 2023

INTRODUCTION

The force‐length relationship of various skeletal muscles indicates that passive force is generally much lower than active force [1]. However, a previous study showed that the whole frog tibialis anterior (TA) muscle has significantly higher passive than active forces within its normal range of motion (Figure 1) [2].

It is known that titin, the giant protein within the sarcomere, is a main contributor to passive force in skeletal muscle at rest [3]. Furthermore, variations in titin isoforms have been related to different passive forces; the shorter the titin isoform, the higher the passive force [3, 4]. Therefore, the high passive force observed in frog TA muscle could result from the expression of a short titin isoform and thus a high titin‐based passive force.

The purpose of this study was to investigate titin isoforms and titin‐based passive force in frog TA muscle. Titin‐based passive force was determined by testing passive force in single skinned muscle fibres in which titin is considered to be the major source of passive force [3].

METHODS

Titin isoforms: Titin isoforms in frog TA and rabbit psoas muscles were determined using agarose strengthened 2% acrylamide SDS‐PAGE gels.

Skinned fibre experiment: Active and passive force‐length relationships were established by stretching skinned frog TA fibres (n=10) to various sarcomere lengths (2.4, 2.6, 2.8, 3.0, 3.2, 3.4 and 3.6 μm), and isometrically activating them after steady state force had been reached.

RESULTS

The frog TA expresses two titin isoforms similar to those expressed by rabbit psoas muscle (3.3 and 3.40 MD).

The active and passive force‐length relationships in TA skinned fibres indicate that passive force is significantly lower than active force on the descending limb of the force‐length relationship (Figure 2).

DISCUSSION AND CONCLUSIONS

Frog TA titin isoforms are not different from those of rabbit psoas, which are known to have relatively low passive [3, 4] compared to active force. Single skinned fibres isolated from frog TA muscle do not produce high passive compared to active forces. Therefore, it seems that titin is not responsible for the high passive force observed at the whole frog TA muscle level. It is likely that the high passive forces produced by the frog TA muscle originate from the extracellular matrix rather than structures within the fibres.

The high passive forces found for the entire frog TA muscle might have implications for the function of the muscle during swimming. Since frogs have large webbed feet, a high passive force in the TA allows frogs to use their feet like human swimmers use passive fins to increase the speed and decrease the energy expenditure of swimming.

Support or Funding Information

CIHR, NSERC, AIHS
Glucocorticoid Receptor Beta Enhances Myoblast Proliferation and Myotube Formation, and Reduces Dexamethasone‐Induced Atrogene Expression
Authors: Marino Joseph S. , Peck Bailey , Sheck Evan , Stroup Steven , Hinson Jennifer , Arthur Susan , Hinds Terry D.
Published: November 2023

Background

Chronic elevations in glucocorticoids from endogenous production or exogenously due to disease treatment, cause skeletal muscle proteolysis and loss of muscle mass. The alpha isoform of the glucocorticoid receptor (GRα) but not the beta isoform (GRβ), has a glucocorticoid binding domain, implicating GRα as the mediator of glucocorticoid‐induced proteolysis. Because GRβ is known to cause glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. To date, there is a significant lack of research on the role of GRβ in the regulation of glucocorticoid‐induced skeletal muscle atrophy.

Purpose

The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and the responsiveness of atrogenes to dexamethasone.

Methods

Mouse GRβ cDNA was overexpressed in C2C12 mouse myoblasts using lentivirus. Changes in cell proliferation were quantified using an MTT assay. Myosin heavy chain staining of day 4 myotubes was used to quantify the effects of GRβ overexpression on indices of myotube formation. Real‐time PCR was used to investigate changes in mRNA expression of glucocorticoid responsive genes and western blot was used to quantify changes in protein expression in response to GRβ overexpression.

Results

GRβ protein expression in C2C12 mouse myoblasts was unaffected by dexamethasone, however, increased in response to insulin. Lentiviral‐mediated GRβ overexpression increased mRNA levels of MyoD and myogenin in confluent myoblast cultures. Furthermore, GRβ overexpression led to an enhanced proliferation of myoblasts associated with reduced mRNA levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Following four days of differentiation, GRβ overexpressing myotubes had increased total number of nuclei within myotubes, nuclei per myotube, the number of myotubes, and a greater fusion index. Lastly, when treated with dexamethasone for 24 hours, GRβ overexpressing myotubes had a blunted MAFbx and MuRF1 atrogene response.

Conclusions

Overexpression of GRβ in skeletal muscle may reduce the catabolic effects of glucocorticoids on skeletal muscle by reducing atrogene responsiveness, primarily causing a state of glucocorticoid resistance. Increasing GRβ levels may stabilize muscle mass and preserve metabolic and physical function during exposure to high doses of glucocorticoids. Future work is warranted to determine the effects of GRβ overexpression in vivo in response to physical, social, and pharmacological models of glucocorticoid excess.
Energy sensing pathways differentially regulate peroxisomes in skeletal muscle vs. liver
Authors: Fuller Scott E. , Worsham Emily A. , Simon Jacob , Gettys Thomas W. , Mynatt Randall L. , Noland Robert C.
Published: November 2023

Peroxisomes in skeletal muscle have the potential to combat lipid‐induced insulin resistance by providing an alternate route for lipid disposal which could reduce buildup of lipotoxic species while concomitantly limiting mitochondrial lipid overload. While much is known about regulation of peroxisomes in liver, there is limited information defining regulation of peroxisomes in skeletal muscle. These studies were designed to gain an understanding of peroxisomal regulation in skeletal muscle. Results indicate peroxisomes in rodent skeletal muscle were virtually unresponsive to ‘traditional’ stimuli that induce peroxisome proliferation in liver, such as peroxisome proliferator‐activated receptor (PPAR) agonists and lipid exposure. Alternatively, peroxisomal markers increased in skeletal muscle in response to dietary methionine restriction and caloric restriction. Interestingly, this response was specific to skeletal muscle as peroxisomal markers in the liver were decreased in response to these dietary manipulations. Since many beneficial effects incurred by caloric restriction are reported to be mediated via activation of energy sensing pathways, we further examined whether AMPK or sirtuin 1 (Sirt1) mediate tissue‐specific peroxisomal regulation. In support of this concept, pharmacological activation of AMPK (AICAR) and Sirt1 (resveratrol) enhanced peroxisomal genes in C2C12 myotubes, but decreased genes in AML‐12 hepatocytes. Initial findings point toward PGC1a as a potential downstream integration point mediating these effects in skeletal muscle. Overall, these findings support a model wherein skeletal muscle peroxisomes are uniquely responsive to energy status signals and interventions that activate these pathways, such as caloric restriction, exercise, cold exposure, etc., may mediate some of their therapeutic action via upregulation of peroxisomes in skeletal muscle.

Support or Funding Information

This work utilized the Genomics Core facilities at PBRC which is supported in part by COBRE (NIH 8 P20‐GM103528) and NORC (NIH 2P30‐DK072476) center grants from the National Institutes of Health. This research was also supported by NIH grants R01DK103860 (R.C.N.), R01DK089641 (R.L.M.), and a pilot and feasibility grant from NORC 2P30‐DK072476 (R.C.N.). Also, S.E.F. is supported by T32 fellowship NIH T32 AT004094.
Increase in perceived stress distinguishes severity of menstrual disruption in young women in response to a 3 month diet and exercise intervention
Authors: Williams Nancy I. , Lieberman Jay , De Souza Mary Jane
Published: November 2023

Low energy availability and psychosocial stress can independently and in combination disrupt reproductive function in mammalian species. However, prospective studies demonstrating both of these effects in humans are rare. We previously showed a causal link between low energy availability and the total number of induced menstrual disturbances (luteal phase defects, oligomenorrhea, and anovulation) in previously sedentary women in response to a 3 month diet and exercise intervention. In the current analysis we tested whether changes in perceived stress (Cohen, 1983) also contributed to the observed menstrual disturbances. Forty previously sedentary women age 18–24 yrs, BMI 51–69 kg/m2, gynecological age 4–15 yrs completed approximately 3 months of a diet and exercise protocol where original group assignment was to either a group that maintained energy balance or one of four groups at varying combinations of diet and exercise manipulated to create low to severe energy deficiency. All subjects ovulated during the baseline cycle prior to the onset of 3 months of supervised exercise (5X per week, moderate to vigorous aerobic exercise 20–75 min) and controlled feeding. Menstrual disturbances were assessed using daily urinary measures of estrone‐1‐glucuronide, pregnanediol glucuronide, and mid‐cycle luteinizing hormone in addition to menstrual calendars. The intervention produced modest weight loss (0–4 kg) and expected increases in body composition and aerobic fitness. As previously reported, the magnitude of the energy deficit predicted the frequency but not the severity of menstrual disturbances such that the greater the energy deficit the greater the sum of all types of menstrual disturbances observed during the three month intervention. In the present analysis, subjects were divided into those who experienced a) no disturbances (NONE; n=14), b) either luteal phase defects and or oligomenorrheic cycles (36 days or greater)(LUT_OLIGO; n=13) or c) at least one anovulatory cycle (ANOV; n=10). The change from baseline to post study in perceived stress was significantly greater in ANOV vs. NONE and LUT_OLIGO: 10.1±3.6, 1.7± 1.7, −0.33± 1.9, respectively (P<0.05; ANOVA and post hoc LSD). All ANOV subjects also experienced either a luteal phase defect or an oligomenorrheic cycle during the intervention, and a higher proportion exhibited either a luteal phase defect or oligomenorrheic cycle at Baseline when compared to NONE and LUT_OLIGO (P<0.05; ANOV and post hoc LSD). These results suggest that increases in perceived stress may contribute to anovulation in the context of an exercise training program that is accompanied by weight loss.

Support or Funding Information

National Institutes of Health Grants RO1‐HD‐39245‐01 (N. I. Williams) and M01‐RR‐10732
Androgen receptor knockout in ovarian theca cells results in altered steroidogenesis in diestrous female mice
Authors: Tram Meilinn , Andrisse Stanley , Wolfe Andrew , Wu Sheng
Published: November 2023

Approximately one out of ten women experience polycystic ovarian syndrome (PCOS). PCOS women exhibit hyperandrogenemia, oligo‐ or anovulation, and polycystic ovarian follicles. Females who exhibit elevated androgen levels have been shown to have reproductive abnormalities. However, it is unknown which target organs are impacted by elevated androgen levels. This study aims to determine the role of androgen receptors (AR) on steroidogenesis and the reproduction of female mice in ovarian theca cells. We utilized a cre‐lox model system to produce ovarian theca cell‐specific androgen receptor knockout (ThARKO) mice. ThARKO mice have dramatically reduced AR expression levels as assessed by both immunocytochemistry and real‐time qPCR. We also investigated age at puberty by assessing the age at vaginal opening and first estrus. There was no difference in the age at vaginal opening between control and ThARKO mice. There was also no significant difference in estrous cyclicity assessed by vaginal cytology. Ovaries of control and ThARKO mice were collected at diestrus and ovarian gene expression was compared in 2 month old mice. During diestrus, we observed an increased mRNA expression of cyp17 compared to control littermates. Cyp17 is the rate limiting step of androgen synthesis. Although fertility was not altered, the AR in theca cells may play a role in steroidogenesis.

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R00 DH056139, P60 DK079637, R01 HD068777
Differential Effects of Menopausal Hormone Formulations on Plasma Orexin A levels in Women of the Kronos Early Estrogen Prevention Study
Authors: Cintron Dahima , Beckman John , Muthuvel Jayandran , Miller Virginia
Published: November 2023

Orexin A (OxA) is a hypothalamic neuropeptide involved in regulation of feeding behavior, metabolism, sleep‐wakefulness and emotion. Plasma levels of this peptide increase with age in both men and women. However, little is known about changes in circulating OxA levels during age‐associated, sex‐ specific hormonal changes, such as menopause, or the effect of menopausal hormone therapy (MHT) on OxA levels. No studies have assessed changes of OxA levels after discontinuation of MHT. The objective of this study was to assess plasma OxA during and three years after cessation of either oral conjugated equine estrogen (o‐CEE, n = 17), transdermal 17β‐estradiol (t‐E2; n= 20), or placebo pills and patches (n = 22) in healthy recently menopausal women (> 6 months < 3 years from their menses) who participated in the Kronos Early Estrogen Prevention Study. The plasma OxA was measured by enzyme‐linked immune absorbent assay prior to randomization to treatment (Baseline) and at 12 months, 24 months, 36 months, 48 months after randomization and at 36 months after cessation of study MHT. Data are presented as median with 25th and 75th percentile. Baseline plasma levels of OxA did not differ among groups (o‐CEE: 3.02 (2.2–4.4) ng/mL, t‐E2=4.09 (3.6–4.5) ng/mL, Placebo= 3.15 (2.5–6.2) ng/mL. During MHT treatment, women randomized to o‐CEE had higher OxA levels when compared to their baseline values at 24, 36 and 48 months of exposure (4.4 ng/mL (2.5–6.3), 4.1 ng/mL(2.5–7.3), 4.2 ng/mL(3.1–6.5), respectively). Changes of OxA levels in the t‐E2 and placebo groups were variable and did not show a consistent trend over the MHT treatment period. Three years after cessation of MHT, only the o‐CEE group showed significantly higher Ox A levels than during the treatment phase of the trial (treatment 3 year discontinuation median – 48 month treatment median =1.369 ng/mL). Our study suggests that the formulation of MHT, in particular o‐CEE (contain metabolites of estrogen) increases plasma OxA. This is the first study to assess Ox A levels for a period of more than 6 months under MHT and after years of discontinuation. If plasma levels of OxA reflect those in the brain, these results may explain in part the beneficial effects of o‐CEE on mood and other menopausal symptoms in women of KEEPS. An increase in OxA driven by o‐CEE could be a mechanism by which MHT helps women regulate energy metabolism and emotional behavior responses.

Support or Funding Information

This project was supported by the Mayo Clinic Office of Health Disparities Research and NIH AG 44170. In addition DC was supported by the CTSA Grant Number TL1 TR000137 from the National Center for Advancing Translational Science, a component of the National Institutes of Health.
Electrophysiological Properties of TRPV1‐Expressing Neurons in the Paraventricular Nucleus of the Hypothalamus
Authors: Butcher Sierra Morgan , Miyata Kayoko , Anwar Imran John , Zsombok Andrea
Published: November 2023

Whole body glucose homeostasis is regulated throughperipheral and central mechanisms, and transient receptor potential vanilloidtype 1 (TRPV1) has been shown to play a role in modulation of glucosehomeostasis. Previously, our datarevealed that liver‐related neurons in the paraventricular nucleus (PVN) of thehypothalamus are regulated by TRPV1‐dependent excitatory neurotransmission, which was diminished in diabetic condition. In this study, a reporter mousemodel was established (TRPV1TdTomato) to identify the location, determinethe cellular properties, and phenotype of TRPV1‐expressing cells. Within the hypothalamus, TRPV1TdTomato cellswere observed in the PVN, dorsomedial, and lateral hypothalamus. Whole‐cell patch‐clamp electrophysiology wasused to establish the cellular properties of TRPV1TdTomato neuronsin the PVN. The average frequency of spontaneousexcitatory postsynaptic currents (sEPSCs) was 2.38 ± 0.6 Hz, with an averageamplitude of 15.83 ± 1.8 pA (n=15). Frequency of miniature EPSCs (mEPSCs) averaged 1.79 ± 0.4 Hz, with an amplitude of 15.66 ± 2.7 pA (n=12). The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs)was 2.37 ± 0.6 Hz, while the average amplitude was 37.61 ± 5.2 pA (n=12). The average frequency of miniature IPSCs (mIPSCs)was 1.44 ± 0.4 Hz, with an amplitude of 31.92 ± 4.6 pA (n=9). The frequency and amplitude of EPSCs andIPSCs recorded from TRPV1TdTomato PVN neurons were similar to thosepreviously recorded from liver‐related and kidney‐related PVN neurons. TRPV1TdTomato neurons fired actionpotentials spontaneously, the firing rate was 1.50 ± 0.44 Hz (n=6) and theinput resistance was 2.06 ± 0.66 GΩ. To determine the phenotype of TRPV1TdTomatoneurons, immunostaining for neuronal cell type markers and an astrocyte marker wasperformed. Our data did not reveal co‐localization with vasopressin, oxytocin, orthe astrocyte marker, suggesting that TRPV1TdTomato cells are notmagnocellular neurons or astrocytes. Insummary, the TRPV1TdTomato mouse model is a useful tool to study therole of TRPV1 in autonomic control; however, further studies are necessary todetermine their phenotype and cellular properties in control and diabeticconditions.

Support or Funding Information

National Institutes of Health (R01 DK099598 for AZ)
Effects of the Neurointermediate Pituitary Lobectomy (NIL) on Pituitary Hormones Blood Level in the Rat
Authors: Organista‐Esparza Alejandro , Viñuela‐Berni Verónica , Tinajero‐Ruelas Manuel , Valdez‐Urias Fernando , Kovacs Kalman , Berczi Istvan , Quintanar‐Stephano Andrés
Published: November 2023

The characterization of the functional state of the NIL animals is important to know all the possible physiological implications of the permanent deficiency of the hormones arginine vasopressin (AVP) and oxytocin (OT). In order to obtain knowledge of the unknown relationships of the effects of NIL on pituitary hormones blood level (GH, PRL, ACTH, TSH, LH, AVP and OT), as well as on urine production, water intake and body weight, groups of Intact Control (IC) and NIL male Wistar rats were compared at 3 and 8 weeks post‐surgery. Hormone levels were assessed by ELISA kits.

Results

NIL caused a transient diabetes insipidus; urine production and water intake returned to normal values between 2 to 4 weeks post‐NIL. AVP and OT serum levels in NIL rats were significantly decreased in 80% and 90% at 3 and 8 weeks post‐surgery. GH and TSH, were significantly decreased at 3 but not at 8 weeks post‐NIL. PRL and ACTH serum levels remained unchanged in both times. Final body weights of NIL animals were 20% less than the IC.

Conclusions

NIL animals are a good experimental paradigm to study the protracted effects of AVP and OT deficiency. It is possible that the decreased GH serum levels NIL‐induced, caused the less gain of body weight of these animals.

Support or Funding Information

Supported by CONACYT‐221262. México (AQS)
A Novel Hypothalamic Action of Adropin: Regulation of Fluid Homeostasis
Authors: Stein Lauren Michelle , Yosten Gina , Samson Willis
Published: November 2023

Adropin, a recently described endogenous peptide encoded by the Energy Homeostasis Associated (Enho) gene, has been reported to be abundantly produced in the liver, smooth muscle and brain. While clinical and transgenic studies have identified adropin as a potent peripheral regulator of energy homeostasis, little is known about its action within the CNS. Therefore, the purpose of our study was to investigate a potential hypothalamic site of action to regulate ingestive behaviors. Male Sprague‐Dawley rats (200–250g) bearing an indwelling cannula in the right lateral ventricle were placed in metabolic cages with free access to lab chow and water (lights out 1800–0600h). During a two‐day habituation period, body weight and ad libitum water and food intakes were recorded. On the day of the experiment, rats were given a 2μl i.c.v. injection of vehicle or vehicle containing 0.3nmol, 1nmol, or 3nmol adropin at 1730h. Cumulative water and food intake was recorded in 30‐minute intervals until 1930h and again the next day at 0800h. While exogenous adropin had only a modest effect on food intake, we observed a profound inhibitory action on anticipatory water intake. Furthermore, after overnight water deprivation male rats administered 3nmol adropin i.c.v. exhibited significantly reduced water intake compared to vehicle treated controls. Importantly, using a deductive ligand receptor matching strategy (AJP 303:R941, 2012) we have identified a putative receptor for adropin. To demonstrate a functional relationship between our candidate receptor and adropin, rats were pretreated with i.c.v. injections of siRNA targeted against either eGFP as a control or our candidate receptor, followed by overnight dehydration and treatment with 3nmol adropin i.c.v. Cumulative water intake was recorded over the next four hours. Compromise of the endogenous candidate receptor significantly abrogated the adropin‐induced inhibition in dehydration‐stimulated water intake. Not only are these studies the first to demonstrate physiologic relevance of adropin as a central regulator of fluid homeostasis, but they provide compelling evidence that the adropin receptor is a formerly orphaned, G protein‐coupled receptor.

Support or Funding Information

NIH RO1 HL121456