bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of intracerebroventricular injection of nesfatin‐1 on autonomic nerve activities in rats and mice
Authors: Tanida Mamoru , Shibamoto Toshishige
Published: November 2023

Nesfatin‐1, one of neuropeptides, reduces appetite and enhances energy expenditure in animals. Previous our experiment showed that intracerebroventricular (ICV) administration of nesfatin‐1 suppressed food intake and stimulated sympathetic nerve outflows to the kidney and the white adipose tissue through the hypothalamic ERK signals (Tanida M., et al., Diabetes 2015). However, it has not been examined whether central nesfatin‐1 affects efferent sympathetic nerve activities to the adrenal gland or the brown adipose tissue regulating cardiovascular function or thermogenesis, respectively, and efferent vagal nerve outflow. Here, we investigated effects of ICV injection of nesfatin‐1 on efferent adrenal sympathetic nerve activity (ASNA) and brown adipose tissue sympathetic nerve activity (BAT‐SNA), and efferent gastric vagal nerve activity (GVNA) in anesthetized rats. In addition, to check species difference in nesfatin‐1 action on autonomic nerves, sympathetic response to ICV injection of nesfatin‐1 was determined in anesthetized mice.

Electrophysiological recordings of autonomic nerve activities in anesthetized rats and mice were performed within the scope of our previous studies (Tanida M., et al., Diabetes 2015, Tanida M., Neuroscience 2013). All animal care and handling procedures were approved by the Animal Research Committees of Kanazawa Medical University. 7–10days after ICV surgery, rats and mice were anesthetized with intraperitoneal injections of a urethane (750 mg/kg) and a‐chloralose (75 mg/kg) mixture. ASNA, BAT‐SNA and GVNA in rats, and sympathetic nerve to the kidney in mice were measured by electrophysiological system.

In the present study, we found that ICV injection of nesfatin‐1 (20, 200 pmol) activated ASNA and BAT‐SNA in dose‐dependent manner while the GVNA was suppressed by nesfatin‐1. In addition, pretreatment with the melanocortin‐3/4 receptor antagonist, SHU9119, attenuated the increase in ASNA induced by nesfatin‐1. These data suggest that hypothalamic nesfatin‐1 stimulates not only renal sympathetic nerve but also sympathetic nerve activities to the adrenal gland and the brown adipose tissue via the hypothalamic melanocortin system to regulate cardiovascular and thermogenic actions. Similar with sympathetic responses of rats to nesfatin‐1, nesfatin‐1 injection to the mice also activated renal sympathetic outflow, indicating that sympathoexcitation of the kidney was also induced in mice.
The Role of Angiotensin Converting Enzyme 1 (ACE1) within the Median Preoptic Nucleus (MnPO) in Chronic Intermittent Hypoxia (CIH) Hypertension
Authors: Faulk Katelynn , Shell Brent , Nedungandi T. Prashant , Cunningham J. Thomas
Published: November 2023

Sleep apnea is associated with a sustained increase in diurnal blood pressure and increased sympathetic tone. CIH simulates the arterial hypoxemia of sleep apnea and produces a sustained increase in blood pressure during the diurnal cycle as well as increased sympathetic activation. Many CNS regions have been shown to contribute to CIH hypertension through increased neuronal activation. FosB, an AP‐1 transcription factor, is associated with increased neuronal activation. Following CIH, FosB positive neurons increase within the MnPO and dominant negative inhibition of MnPO FosB blocks the sustained, diurnal component of CIH hypertension. PCR array analysis following a dominant negative inhibition of FosB within the MnPO during CIH identified several candidate AP‐1 regulated genes that may contribute to the sustained hypertension seen in CIH. ACE1, which converts angiotensin I to angiotensin II, was increased within the MnPO following CIH and this effect was blocked by inhibition of FosB. Increased expression of ACE1 through FosB activation within the MnPO may provide a possible mechanism by which downstream regions, such as the Paraventricular Nucleus (PVN), are activated to sustain diurnal hypertension. Our hypothesis is that CIH increases the expression of ACE1 in MnPO neurons that project to the PVN and that this increase in expression is due to a direct interaction between FosB and the ACE1 gene. We first tested this hypothesis by microinjecting isoflurane anesthetized adult male rats with 200nl of a retrograde tracer, flourogold, unilaterally into PVN. Rats were then exposed to CIH for 7 days through 3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day (0800–1600 h). Controls were only exposed to room air. Mean arterial blood pressure (MAP) was recorded using radio telemetry. Forebrains were fixed through 4% paraformaldehyde perfusion and later processed for ACE1 and FosB immunohistochemistry. In separate groups, we performed a Chromatin Immunoprecipitation (ChIP) assay from the MnPOs of rats following 7 days of CIH treatment. The MnPOs were combined from 2 rats within the same treatment group (CIH or normoxic) in order to get sufficient amount of DNA. ChIP assays were performed with ChIP grade antibodies against FosB and a nonimmune IgG to control for nonspecific antibody binding. MnPO neurons expressing ACE1 and the fluorogold tract tracer significantly increased colocalization with FosB expression following CIH as compared to normoxic controls (P<0.05). FosB also significantly increased association with the ACE1 gene within the MnPO following 7 days of CIH as compared to the normoxic controls (P<0.05). These results demonstrate that FosB and ACE1 are co‐expressed in PVN‐projecting MnPO neurons following CIH and increased ACE1 expression associated with CIH is mediated by FosB.

Support or Funding Information

P01 HL088052
Increased Sympathetic Nerve Activity Due to Relaxin in the Subfornical Organ Is Mediated by RXFP1 Receptors on Astrocytes and Ultimate Activation of Projection Neurons to the Paraventricular Nucleus of the Hypothalamus in Both Female and Male Rats
Authors: Coldren K. Max , Hasser Eileen M. , Heesch Cheryl M.
Published: November 2023

Endogenous relaxin (RLX), an ovarian hormone which is elevated in pregnancy, contributes to peripheral vasodilation in pregnant women and rats. Recent clinical trials in both male and female hospitalized acute heart failure patients (the majority of whom were also receiving blockers of the renin‐angiotensin system) indicate that exogenous administration of human recombinant RLX‐2 (i.v., 48 hrs) relieved dyspnea (due to reductions in pulmonary edema) within hours and reduced post‐discharge mortality at 180 days. In both males and females, RLX receptors are present in the vasculature, heart, and brain. Vascular actions of RLX are inhibitory and mediated through nitric oxide mechanisms, while central nervous system actions of circulating RLX are generally excitatory, mediated by angiotensin II mechanisms, and dependent on RLX receptors (RXFP1) in brain regions devoid of a blood brain barrier. Previously we demonstrated in anesthetized arterial baroreceptor denervated female rats that microinjection of synthetic human RLX‐2 into the subfornical organ (SFO, 0.77 pmol in 50 nl) produced sustained increases in lumbar sympathetic nerve activity (8 ± 3%) and mean arterial pressure (MAP, 26 ± 4 mmHg). Intra‐carotid artery (ica) infusion of a sub‐pressor concentration of RLX (155 pmol/ml/hr; 1.5 hr) increased heart rate (+50 ± 5 bpm) and activated neurons (increased Fos‐IR) in the SFO and in spinally‐projecting (19 ± 2%) neurons in the PVN. We now demonstrate that RLX receptors in the SFO are distributed in an annular pattern dorsal and lateral to activated (Fos‐IR) neurons. In both male and female rats, RLX receptors in the SFO were not located on PVN‐projecting neurons nor on microglia (Iba1‐IR). Astrocytes in the SFO are the major source of angiotensinogen and RLX receptors were associated with astrocytic cell bodies (S100B‐IR) and processes (GFAP‐IR) which were in close apposition to PVN projecting neurons. These data provide an anatomical substrate for a role of circulating RLX to modulate SNA through a mechanism possibly dependent on local formation of angiotensin II in the SFO. We speculate that RLX administration to decompensated heart failure patients could produce central sympathoexcitatory effects, but that continued treatment with angiotensin converting enzyme inhibitors or AT1 receptor antagonists in clinical trials may have blunted these untoward effects and contributed to positive outcomes.

Support or Funding Information

NIH R01‐HL091164
Quantification of Cardiac Baroreflex Sensitivity during Autonomic Stimulation
Authors: Sharma Purnima , Mavai Manisha , bhagat Om lata , Murugesh Meghna , Sircar Sabyasachi
Published: November 2023

Background

The cardiac baroreflex system plays a major role in the short‐term regulation of the blood pressure. The baroreflex function as an interface between the central nervous and cardiovascular systems and in consideration of its clinical significance, the quantification of Baroreflex function is of high relevance. Its functional properties are strongly influenced by the parasympathetic system. In this study, we quantified Baroreflex sensitivity (BRS) during slow deep breathing (SDB) and cold pressor test (CPT).

Material and Methods

In 14 healthy volunteers’ (7 males and 7 females; mean age 17–21 yrs) baroreflex sensitivity (BRS) was quantified by applying sequence analysis based on continuous recording of blood pressure and heart rate. The BRS was quantified during slow deep breathing at 6 breaths/minute, cold pressor test at 5°C temperature for maximum 5 minutes.

Results

A significant increase in BRS up sequences was observed during the SDB as compared to the rest (18.57 ± 11.84 vs 31.52 ± 11.69; p = 0.01). Cold pressor test did not show any significant difference in the BRS up sequences compared to the rest (18.57 ± 11.84 vs 18.34 ± 9.89; p > 0.05). A significant lower BRS up sequence was observed during CPT as compared to the rest (18.34 ± 9.89 vs 31.52 ± 11.69; p = 0.01). A significantly higher BRS down sequences were observed during SDB as compared to the CPT (13.64 ± 8.03 vs 25.68 ± 8.44; p = 0.04). A significantly higher BRS all sequence was observed during the SDB as compared to the CPT (17.37 ± 8.57 vs 28.28 ± 7.2; p = 0.02).

Conclusion

The present study revealed a substantial increase in baroreflex sensitivity during slow deep breathing, which constitutes a widely accepted measures of parasympathetic tone.

Support or Funding Information

Indian Council of Medical Research (ICMR)
Reduced bone marrow adrenergic receptor signaling modulates PVN inflammatory factors
Authors: Ahmari Niousha , Schmidt Jordan T , Martyniuk Christopher T , Zubcevic Jasenka
Published: November 2023

INTRODUCTION

Neurogenic hypertension is characterized by neuroinflammation and increased sympathetic nerve activity (SNA) to the periphery, including the bone marrow (BM). The BM SNA governs diurnal regulation of the BM hematopoietic cell turnover, with inflammatory cells typically released during the active period. We have previously shown that chronic increase in the BM SNA leads to elevation of inflammatory factors and release of inflammatory cells in angiotensin II (Ang II) hypertension (HTN). Moreover, extravasation of the BM‐derived inflammatory cells to the brain cardioregulatory regions, including the paraventricular nucleus (PVN), contributes to neuroinflammation and thus the pathophysiology of HTN. Therefore, we hypothesize that reduction in the effects of SNA on BM inflammatory cells will result in suppression of the inflammatory profiles in BM and PVN.

METHODS

A novel chimeric BM mouse was created by reconstituting sub lethally‐ irradiated C57BL/6J mice with whole BM cells from adrenergic beta 1 and beta 2 receptor knock out (AdrB1.B2KO) mice (Jax Labs). Following recovery, relative expression levels of Adrb1 in circulating mononuclear cells were assessed using quantitative RT PCR, in order to confirm the success of irradiation/reconstitution. Total RNA from PVN and BM was isolated and purified, and gene expression profiles were determined using a mouse 60K Agilent array. Differentially expressed gene networks were determined in each tissue using Pathway Studio (Elsevier).

RESULTS

There were 8563 and 477 differentially expressed probes in the BM and PVN of AdrB1.B2 KO mice, respectively (FDR <0.05). Upon further investigation into the 53 gene networks differentially expressed in the BM and PVN in the AdrB1.B2 KO mice compared to CNT, it was determined that 13 pathways were related to the immune system. In the BM alone, 41 gene networks associated to immunity were observed in the KO chimera vs CNT (p <0.05). This included a decrease in gene network that included T cell adhesion and leukocyte accumulation gene networks (P<0.05). In terms of specific transcripts associated with these networks, CD4 and CD8a expression was downregulated by 1.3 fold (p<0.007), and 2.6 fold (p<0.01) respectively. In the PVN, there was a 1.3 fold downregulation of IL17 receptor alpha and beta (p<0.015), as well as a decrease in CCL2 expression (p<0.018).

CONCLUSION

In summary, reduction in responsiveness of the BM hematopoietic cells to SNA is associated with a reduction of inflammatory mediators in both the BM and PVN. Therefore, BM cells’ ability to respond to SNA stimulation can affect both central and peripheral inflammatory responses. This is expected to have significant implication in neurogenic HTN, characterized by both increased sympathetic drive and exaggerated immune responses.
Do Standard Laboratory Practices Lead to Obese Zebrafish Populations?
Authors: Watts Stephen Andrew , Dennis Lacey , Fowler Lauren Adele , Williams Michael Britt , Yuan Yuan , Barry Robert Jeff , Farmer Susan , D'Abramo Louis R , Powell Mickie Lynn
Published: November 2023

The value of the zebrafish model is well established and its widespread utility is without question in the study of early development, environmental science, and translational approaches to human disease onset and progression. Outcome variability due to the absence of nutritional standardization within and among laboratories remains, and can be magnified due to a minimal understanding of the potential role of nutrition. The community continues to use embryos and adults derived from stocks in which the nutritional history is not defined, thus, direct or indirect effects of nutrients on outcomes cannot be evaluated. In this study we compared the growth demographics and body composition associated with feeding common commercial diets (closed formulation) and a purified reference diet to zebrafish.

Twenty‐one day old juvenile fish were fed one of six diets (commonly used by the majority of laboratories in the US as determined by the Zebrafish Husbandry Association). Each diet treatment contained nine 2.8 L tanks, each containing 13 zebrafish (n=117 ind/treatment). Fish were fed twice daily (09:00 and 17:00 hr) at a ration of >5% body weight per day (based on the fastest growing treatment). Rations were adjusted biweekly. Weight gain varied among the diets, with 14 week mean weights varying among treatments from 670 to 950 mg fresh weight. Lipid content of males and females (ovary removed) ranged from 22 to 42 and 18 to 45% dry matter, respectively. As compared to fat content estimated in wild fish populations, the levels of body lipid reported here would suggest these lab‐reared zebrafish have excessive body fat that is relative to that observed in human obesity. These data support recent metabolic literature indicating zebrafish are excellent models for the study of obesity and related morbidities. Variations in growth and body composition analysis in response to common laboratory diets and practices highlight the problems associated with the lack of nutritional information and standardization of diets in the zebrafish research community. There is a critical need for the development of standardized diets and feed management strategies. This should be a high‐priority community effort among laboratories with support from ASN and NIH. The long‐term goal of nutrition research in zebrafish will be to identify the daily nutritional requirements and to develop appropriate standardized reference and open formulation diets for the zebrafish community.

Support or Funding Information

Research and animal care supported in part by the American College of Laboratory Medicine and the Aquatic Animals Research Core within the UAB NORC (P30DK056336).
Exercise responses in aged rats selected for High versus Low training response
Authors: Sepe Joe , Koch Lauren G. , Britton Steven L. , Diffee Gary
Published: November 2023

Exercise training improves aerobic capacity, but there are a number of gaps in our understanding of the exercise training response: 1) There is a wide variability in the response to training among individuals (both human subjects and rodents), but little is known about the mechanisms underlying this variability in training response. 2) It is unclear to what extent aging affects the ability to adapt to endurance training. To address these questions, we used a contrasting animal model system in which rats were artificially selected for 19 generations into High Response Trainers (HRT) versus Low Response Trainers (LRT), based on their response to aerobic treadmill training. Exercise capacity (as measured by time to exhaustion, EX‐TIME) and VO2max were measured in 22 month old rats and then animals were divided into Sedentary (SED) vs. Exercise (EX) groups. EX animals were trained 5 days a week for 3 months, with speed and duration gradually increased to the point that animals were running at 14 m/min for 45 min. At the end of the 3‐month training period, exercise capacity and VO2max were again measured in all animals. Mean EX‐TIME and VO2max values significantly (p <0.05) declined in SED animals from Pre‐training (PRE) to Post‐training (POST). EX‐ TIME declined by 35% in POST compared to PRE, while VO2max declined by 26% in POST vs. PRE. In the EX animals there was no significant difference in PRE vs. POST EX‐TIME or VO2max mean values in the LRT group, while in the HRT group, there were significant (p <0.05) increases in EX‐TIME (POST values 147%) of PRE) and VO2max (POST values 131% of PRE). To determine the mechanism of this variability in exercise training response in old animals, we correlated individual animal's improvement in EX‐TIME to measures of their cardiac function using echocardiography and measures of expression of genes related to mitochondrial biogenesis in skeletal muscle. Improvements in EX‐TIME were well correlated with improvements in VO2max. While EX training significantly (p <0.05) improved mean Fractional Shortening (FS%) and Ejection Fraction (EF%) in HRT compared to SED, ( EF% increased by 19% compared to SED; FS% increased by 31%), in individual animals these values did not correlate with improvements in EX‐TIME. However, increases in expression of a number of genes related to mitochondrial biogenesis and PGC‐1alpha signaling were well correlated with improvements in EX‐TIME. These results show that 1) aging animals retain the ability to respond to endurance training with improvements in VO2max and exercise capacity, but a differential to respond between HRT and LRT animals remain, and 2) changes in expression of genes related to mitochondrial biogenesis appear to underlie individual variation in the response to exercise training.

Support or Funding Information

Supported by NIH AG 030423
Arginine and Aerobic Training Prevent Impairment in Endothelial Function in Rats Under High Risk to Develop Metabolic Syndrome
Authors: Medeiros Renata Frauches , Gaique Thaiane Gadioli , Bento‐Bernardes Thais , Britto Fernanda Carla Ferreira , Motta Nadia Alice Vieira , de Jesus Oliveira Karen , Nóbrega Antonio Claudio Lucas
Published: November 2023

It is not elucidated if aerobic training and arginine supplementation can have similar protective effects in individuals with constant exposure to the triggering agent of the metabolic syndrome, with the presence of subclinical alterations, but without the presence of cardiovascular disease. So the purpose of this study was to investigate the effect of arginine supplementation and aerobic training on vascular reactivity of rats under high risk of metabolic syndrome development. Wistar rats, males, adults were, initially, divided in two groups Control group (C; drinking water) and Fructose group (F; water with 10% fructose), after two weeks the F group was divided into four others groups: Fructose group (F), that continued with the same treatment, Fructose Arginine group (FA), Fructose Training group (FT) and Fructose Training Arginine group (FTA) same as F group, adding aerobic training on a treadmill and/or arginine supplementation by gavage (800mg/kg BM/day), in the last 8 weeks. There were no differences in body mass gain and caloric intake between groups (P>0.05). It was observed higher serum insulin in F group compared to control group after two (P=0.046) and ten weeks (P=0.014), while it was not observed any difference between the other groups compared to C group. In relation to serum triacylglycerol levels, after two weeks, there was no difference (P=0.154) between C and F groups. After ten weeks the F group presented higher triacylglycerol concentration (P<0.01) compared to C group and the training normalized this increased, however arginine supplementation had no effect on this parameter. The malondialdehyde serum concentration, an oxidative stress marked, had the same pattern as the triacylglycerol. Concerning vascular reactivity, there was no difference after two weeks of treatment. While at the end of protocol, the F group exhibited increased vasoconstrictory reactivity (P<0.01) to phenylephrine when compared to C and to trained groups (FT and FTA). The F group also presented an impaired endothelial vasodilator response to acetylcholine compared to other groups and the arginine supplementation and the aerobic training were able to prevent this impaired response. Thus aerobic training prevents the metabolic alterations, the hypereactivity to phenylephrine and the endothelial damage of rats with high risk to develop metabolic syndrome, while arginine supplementation was capable only to prevent the endothelial damage in this experimental model, with no potentiating effect.

Support or Funding Information

CAPES, CNPq, FAPERJ e FINEP.
Inhibition of UPRER Does Not Impair Signaling to Mitochondrial Biogenesis During Chronic Contractile Activity
Authors: Oliveira Ashley N , Memme Jonathan M , Hood David
Published: November 2023

Cellular stresses that perturb the protein folding environment can be detrimental to cell health and may provoke apoptosis. In order to combat these stresses, the cell is equipped with compartment‐specific mechanisms to help regain homeostasis: the mitochondrial (UPRmt) and endoplasmic reticulum (UPRER) unfolded protein responses. Acute exercise is capable of eliciting the UPR, however the role of the UPR in chronic exercise‐induced adaptations is still unclear. Exercise training can be modeled by direct stimulation (6V, 10Hz; 3hrs/day) of the peroneal nerve in Sprague Dawley rats to induce chronic contractile activity (CCA) for 7 days. We can model endurance training in one hindlimb, while the other serves as an internal control. Previously, we have shown that UPRER signaling is upregulated during the early phases of CCA, as shown by 1.5–4 fold increases in BiP, CHOP and XBP1 spliced mRNA. We then sought to investigate the role of the UPRER in mitochondrial adaptations by inhibiting UPR signaling with tauroursodeoxycholic acid (TUDCA). TUDCA treatment during 7 days of CCA attenuated CHOP activation by 63%, suggesting effective impairment of UPRER signaling. Despite this, mitochondrial biogenesis (MB) was not reduced by TUDCA treatment during CCA, evidenced by comparable 22–24% increases in COX activity, a measure of mitochondrial content, after 7 days of CCA. Since the vast majority of proteins that are required for MB are nuclear‐encoded and require import into the organelle, this could imply an important role for protein import in UPRmt function. Tim23, a major translocase of the inner membrane, increased by an average of 23% as early as 1 day of CCA, suggesting a greater potential for the transport of nuclear‐transcribed proteins into the matrix. If protein import into the matrix is increased, this could be accompanied by a greater protein folding/handling capacity. Using this CCA model we can further investigate the role of protein import and its relationship with the UPRmt in potentially mediating mitochondrial adaptations to chronic exercise.

Support or Funding Information

Supported by NSERC.
Effects of Interval and Continuous Exercise Regimens on Aquaporin‐1‐mediated Osmotic Fragility of Erythrocyte under Hypoxic Stress
Authors: Huang Yu‐Chieh , Wang Jong‐Shyan
Published: November 2023

Physical exercise or hypoxic exposure influences erythrocyte susceptibility to osmotic stress. Aquaporin‐1 (AQP1) channel facilitates water transport in erythrocytes. This study investigated how aerobic interval training (AIT) and moderate continuous training (MCT) affect osmotic stress‐mediated rheological function and AQP1 activity of erythrocytes under hypoxic stress. Thirty healthy sedentary males were randomized to engage either AIT (3‐minute intervals at 40% and 80% VO2max, n=15) or MCT (sustained 60% VO2max, n=15) for 30 minutes/day, 5 days/week for 6 weeks. Erythrocyte rheological responses to HE (100W under 12%O2 for 30 minutes) were determined before and after various regimens. The results showed that acute HE increased osmotic fragility and decreased deformability of erythrocytes, as well as, depressed erythrocyte AQP1 activity indicated by increased HgCl2‐induced instability of erythrocyte membrane under hypotonic conditions. Following 6 weeks of the intervention, the AIT group exhibited higher maximal work‐rate and VO2max than the MCT group. Moreover, both AIT and MCT diminished the extents of enhanced osmotic fragility, reduced deformability, and AQP1 activity of erythrocytes caused by HE. Hence, we conclude that AIT is superior to MCT for enhancing aerobic capacity. Moreover, either AIT or MCT effectively alleviates the impairments of erythrocyte rheological characteristics and AQP1 function evoked by HE.
Exercise Acclimation but not Thermal Area Alters the Cytokine Response to Exertional Heat Stroke
Authors: King Michelle Ann , Clanton Thomas
Published: November 2023

Our group has previously established a model of exertional heat stroke (EHS) that displayed a unique cytokine profile, where cytokines classically involved in the innate immune response were blunted and moved earlier in time compared to passive heat stroke (PHS) models. Limitations in the experimental design may have inadvertently led to this finding. Our objective was to account for these limitations and identify the source of the blunted cytokine response. Acclimation sessions (mild training) and decreased thermal area (TA) were identified as potential sources of cytokine suppression. To explore the effect of acclimation, C57/BL6 mice (n=6) did not receive enrichment or acclimation prior to EHS. Interestingly, exercise acclimation sessions clearly increased the circulating concentrations of IP‐10, KC, MCP‐1, and MIP‐1β (P<0.05), but had no effect on other cytokines. Typical EHS mice experience a TA of »146 (°C x minutes), whereas mice exposed to a “classic” PHS model are exposed to a TA of 409 (°C x min). To control for the influence of the duration of heat exposure, six mice underwent PHS at elevated ambient temperature, designed to match the TA in EHS. Circulating cytokines in this accelerated PHS experiment were similar to classic PHS and did not resemble EHS. In conclusion, shortened thermal area does not account for altered cytokine responses in EHS. However, exercise acclimation changes the magnitude of specific cytokine responses to EHS. This effect may have implications for the positive effects of exercise conditioning or stress reduction on the incidence of heat illness.
ROLE OF CENTRAL TRPV4 IN THE ACTIVATION OF HEAT LOSS RESPONSES IN WISTAR RATS
Authors: Bicego Kenia C , Scarpellini Carolina da S. , Almeida Maria Camila , Gargaglioni Luciane H.
Published: November 2023

In vitro results and studies with knockout mice for TRPV4 have suggested that this receptor is activated by warm temperatures. Recently, our group has described that peripheral TRPV4, activated by ambient temperatures (Ta) between 26 and 30oC, stimulates warmth‐defence responses in non‐genetically modified rats; however no data was available about the role of hypothalamic TRPV4 in thermoregulation. Then, the aim of this study was to verify if central TRPV4 in the medial preoptic area (MPA) plays a role in the heat loss activation in rats. To this end, body temperature (Tc), metabolic rate (VO2) and heat loss index (HLI) were measured at Ta of 21 and 28°C after TRPV4 in MPA were antagonized by HC‐067047 (0.5, 5 and 25ng/50nL) . The antagonism of central TRPV4 caused an increase in the Tb of animals exposed to 28°C by approximately 1°C (5ng/50nL; p< 0.001), but those animals exposed to 21°C had their Tb increased only by ~0.4°C (5 and 25ng/50nL; p< 0.001). At 28°C, VO2 (p= 0.012), but not HLI, was also increased by the antagonist. Our results suggest, for the first time, that central TRPV4 plays a role in autonomic heat loss responses in rats. Moreover, this action appears to be dependent on peripheral activation, i.e., when peripheral TRPV4 are not activated, the central ones have a less evident role in the stimulation of heat loss.

Support or Funding Information

FAPESP 2011/19131‐2 and 2015/04849‐6
Molecular mechanisms behind the accumulation of lipid that occurs after skeletal muscle injury
Authors: Gumucio Jonathan , Rittman Danielle , McDonagh Brian , Mendias Christopher
Published: November 2023

Myosteatosis is the accumulation of lipid that occurs after skeletal muscle injury or in certain chronic neuromuscular or metabolic diseases. Muscle fiber atrophy and fibrosis often accompany myosteatosis. The amount of lipid accumulation is negatively correlated with skeletal muscle functional capacity, but the causes of myosteatosis are unknown. To gain a greater understanding of the ontogeny of myosteatosis, we induced a severe injury to the rotator cuff musculature in rats, isolated tissues 10, 30 or 60 days after tear, and used a combination of RNA sequencing and shotgun lipidomics to identify global changes in gene expression and lipid content in injured muscles. The RNA sequencing data and shotgun lipidomics results were then analyzed by Ingenuity Pathway Analysis (IPA, Qiagen) software to determine the biochemical pathways involved in myosteatosis. Samples were also prepared for electron microscopy and single muscle fiber contractility. Following muscle injury, there was a time dependent increase in total lipid content, largely due to a dramatic rise in triacylglyceride species. Interestingly, genes related to lipid synthesis (DGAT1, DGAT2) and lipid breakdown (LPL, HSL) were largely downregulated over time. IPA of RNASeq data predicted increased mitochondrial dysfunction and oxidative stress, and decreased lipid oxidation to be highly associated with myosteatosis at all time points. Genes related to lipid utilization and mitochondrial function (CPT1/2, SDH, COXIV) were also decreased with time. From electron micrographs, there was increased subsarcolemmal mitochondria at all time points after injury and dramatic sarcomere streaming at 30d and 60d compared to controls. Additionally, there was an increase in carbonylated proteins at 60d. At 30 and 60d, IPA predicted increased inflammatory pathways in chronically torn rotator cuff muscles compared to controls, and there was an overall increase in inflammatory gene markers (IL6, IL1b, COX1) at all time points. These changes corresponded to a time dependent decrease in muscle fiber specific force production, or muscle fiber maximum isometric force normalized to cross sectional area. Combined, these data suggest that the accumulation of lipid that occurs after muscle injury is likely due to a decrease in lipid utilization and breakdown rather than an increase in lipid synthesis. Sustained mitochondrial dysfunction and oxidative stress may result in the increased lipid infiltration and atrophy of muscle fibers over time. While further investigation is necessary, this study provided important insight into the development of myosteatosis and identifying potential therapeutic targets to improve muscle regeneration.

Support or Funding Information

This work was supported by NIH grants (R01AR063649 and F31AR065931)
Inhibition of Toll‐like Receptor 4 does not prevent palmitate‐induced endoplasmic reticulum stress in cultured skeletal muscle cells
Authors: Perry Ben Douglas , Rahnert Jill A , Zheng Bin , Price Russ
Published: November 2023

Chronically elevated circulating saturated fatty acids (SFA), as seen in Type 2 Diabetes Mellitus, impairs insulin sensitivity, protein synthesis, and upregulates proteolytic pathways in skeletal muscle. The SFA‐induced impairment of muscle protein synthesis is due, in part, to increased endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response. However, the upstream receptor(s) that initiate ER stress in response to SFA in skeletal muscle are not well delineated. This study investigated the role of TLR4, which is activated by SFA and induces pro‐inflammatory signaling, on markers of ER stress and protein synthesis signaling in cultured mouse muscle cells (C2C12). C2C12 myotubes were treated with either vehicle, a TLR4‐specific ligand (lipopolysaccharides; 100 ng/mL), palmitate (SFA; 500 μM), or a combination of palmitate and TLR4‐specific inhibitor (TAK‐242, 1 μM). After 24 hours, cells were harvested and analyzed for markers of ER stress and protein synthesis using RT PCR and western blotting. Palmitate treatment substantially increased the levels of CHOP, spliced XBP1, and ATF 4 mRNAs as well as the protein expression of p‐PERK/PERK and p‐eIF2α/eIF2α. The palmitate/TLR4 inhibitor treatment unexpectedly elevated both CHOP mRNA and protein expression of p‐eIF2α/eIF2α to levels greater than palmitate alone. Compared to control, the p‐p70S6k/p70S6k ratio was reduced to a similar extent with both palmitate and palmitate/TLR4 inhibitor treatments. These data suggest that in conditions involving lipotoxicity, activation of TLR4 in skeletal muscle is not responsible for palmitate‐induced ER stress. Instead, activation of TLR4 may induce signaling which is protective during heightened ER stress in muscle.

Support or Funding Information

Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit I01BX001456
Eutherian BetaM, a Muscle‐Specific Integral Protein of Inner Nuclear Membrane, is Implicated in Regulation of MyoD Expression
Authors: Modyanov Nikolai , Pestov Nikolay , Ahmad Nisar
Published: November 2023

Vertebrate X‐chromosome ATP1B4 genes represent a rare instance of the orthologous gene co‐option that radically changed functions of encoded BetaM proteins during evolution. In lower vertebrates, BetaM is a subunit of Na,K‐ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through radical changes in the structure of its N‐terminal domain gained entirely new properties as a skeletal and atrial cardiac muscle‐specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. BetaM expression is strictly temporally regulated being the highest in late fetal and early postnatal myocytes. Here we show that expression of exogenous BetaM in cultured muscle cells up‐regulates expression of endogenous MyoD, a master regulator of myogenesis, both at transcriptional and translational levels. We determined that eutherian BetaM associates with nuclear transcriptional co‐regulator Ski‐interacting protein (SKIP) and both are identified by ChIP assays as components of MyoD transcriptional complex in vivo in rat neonatal skeletal muscle. Presence of BetaM was detected in the DRR but not in PRR and CE regions of the MyoD promoter. According to the results of Electrophoretic Mobility Shift Assays (EMSA) BetaM‐containing transcriptional complexes are bound to specific E‐box4 and CArG elements of the DRR region. Deletion of either E‐box4 or CArG or both in DRR luciferase reporter abolished its activation by BetaM, These findings demonstrate that BetaM is a novel eutherian‐specific transcriptional regulator which is involved in the regulation of MyoD expression during muscle development. Moreover, structural and functional alterations acquired through ATP1B4 gene co‐option and a unique pattern of expression argue that eutherian‐specific BetaM functions are essential, even might be necessary for survival of placental mammals in natural conditions, and that they provide evolutionary advantage.

Support or Funding Information

Supported by Department of Physiology & Pharmacology, University of Toledo College of Medicine.
Intramuscular NF‐κβ Signaling in Response to Resistance Exercise and Recovery in Untrained Males
Authors: Townsend Jeremy , Stout Jeffrey , Jajtner Adam , Church David , Beyer Kyle , Oliveira Leonardo , Fukuda David , Hoffman Jay
Published: November 2023

The nuclear factor kappa B (NF‐κβ) signaling cascade is responsible for mediating stress‐activated catabolic effects of TNF‐α downstream of the TNF receptor. NF‐κβ transcription factor remains inhibited in the cytosol of the muscle and can be stimulated for translocation and transcription by a variety of external stimuli, most notably by pro‐inflammatory cytokines and oxidative stress. Nevertheless, currently there is a gap in the literature with regard to the time course effect of NF‐κβ signaling following acute resistance exercise in humans.

PURPOSE

To observe the effects of an acute lower‐body resistance exercise protocol and subsequent recovery on intramuscular NF‐κβ signaling.

METHODS

Twenty‐eight untrained males (22.3 ± 3.2 y, 1.7 ± 0.1 m, 81.1 ± 15.5 kg) were assigned to either a control (CON; n=11) or exercise group (EX; n=17) and completed a lower‐body resistance exercise protocol consisting of the back squat, leg press, and leg extension exercise. Skeletal muscle microbiopsies were obtained from the vastus lateralis pre‐exercise (PRE), 1‐hour (1HR), 5‐hour (5HR), and 48‐hours (48HR) post‐resistance exercise. Multiplex signaling assay kits (EMD Millipore, Billerica, MA, USA) were used to quantify the total protein (TNFR1, c‐Myc) or phosphorylation status of proteins (IKKa/b, NF‐κβ, IkB) specific to apoptotic signaling pathways using MAGPIX® (Luminex, Austin, TX, USA). Mann Whitney U analysis was used to determine the effects of the exercise bout on intramuscular signaling at each timepoint. Additionally, change scores were analyzed by magnitude based inferences to determine a mechanistic interpretation.

RESULTS

Mann‐Whitney U analysis revealed that resistance exercise increased total c‐Myc at 1H (p=0.001) and tended to increase with exercise (p=0.053) at 5H. A trend for an increase in IkB phosphorylation was observed at 5H (p=0.053). There were no other significant differences observed. Magnitude based inferences revealed a “Very Likely” increase in total c‐Myc from PRE‐5H and a “Likely” increase in IkB phosphorylation from PRE‐5H.

CONCLUSION

It appears that c‐Myc transcription factor and phosphorylation of IkB are elevated following acute intense resistance exercise in untrained males. These data may indicate that NF‐κβ signaling plays a role in skeletal muscle regeneration following resistance exercise in young untrained males.
Methylsulfonylmethane (MSM) Treatment Enhances C2C12 Wound Closure and Protects Cells from Oxidative Stress
Authors: Touchberry Chad D. , Von Schulze Alex , Amat‐Fernandez Clara , Lee Harold , Chow Ying , Wetmore Lori A
Published: November 2023

MSM is known to provide anti‐inflammatory and anti‐oxidant effects in both animals and humans. Because both exercise and chronic disease have the potential to induce inflammation and oxidative stress in skeletal muscle, MSM may be a novel therapy to counter the negative consequences of these stressors on skeletal muscle function. Recent evidence has shown that MSM supplementation in human subjects can increase serum MSM concentrations to 2–3 mM. These serum concentrations of MSM have been associated with reduced muscle pain and reduced markers of muscle damage (creatine kinase) following damaging exercise. Therefore, MSM may protect muscle against damage or favorably influence the adaptive capacity of skeletal muscle under stress. However, it is not known if MSM induces this protection by directly acting on skeletal muscle. Therefore, our objective was to determine if MSM may provide cytoprotection from oxidative stress, or enhance the recovery processes in C2C12 myoblasts. We found that treating C2C12 myoblasts with increasing concentrations of MSM (0–200 mM) for 24, 48 and 72 hours decreased cell metabolism (via Alamar Blue) at high concentrations (100 & 200 mM); however this decline was not associated with increased cell death (via Trypan Blue). Flow cytometry analysis showed MSM treatment (200 mM) trapped myoblasts in G1 phase (+51%) and reduced the number of cells moving into S phase (DNA synthesis; −38%) or G2+M phase(mitosis; −43%) of cell division. MSM had no effect on C2C12 differentiation (d1, d4, d6). High concentrations of MSM (100 & 200 mM) were able to reduce cell death (−36% & −89% respectively) induced by oxidative stress (tert‐butyl hydrogen peroxide; 20 uM). Because heat shock proteins (HSPs) are known to be cytoprotective, we determined if increasing concentrations of MSM induced HSP expression; however, we found no changes in HSP70 and HSP27 expression. Lastly, lower concentrations (12.5 mM) of MSM enhanced wound closure (+35%) from a scratch assay 24 following treatment, while higher concentrations (200 mM) were associated with an impaired closure rate (−51%). While more applied research is needed, these findings suggest that MSM may directly alter the skeletal muscle response to stress and injury. Therefore, MSM supplementation may serve as an ergogenic aide to protect skeletal muscle from injury.

Support or Funding Information

Funded by Bergstrom Nutrition to CDT
A Role for Titin During Doublet Potentiation of Fast and Slow Twitch Muscles
Authors: Monroy Jenna A , Bailey Elizabeth , Nishikawa Kiisa
Published: November 2023

Doublet potentiation is the increase in muscle force as a result of the addition of a single pulse, or doublet, at the beginning of a low‐frequency stimulus train. The force output is higher in amplitude, generated at a faster rate, persists throughout activation. However, current muscle models fail to accurately predict muscle force during doublet potentiation. Recent work suggests that the elastic protein, titin, may play a role. In this study, we investigated the role of titin in doublet potentiation by using the muscular dystrophy with myositis (mdm) mouse, which is characterized by a deletion in the N2A region of the titin gene. Previous research on the winding filament hypothesis suggests that the N2A region of titin binds to the thin filaments upon activation, which increases titin stiffness and total muscle force. We hypothesized that the absence of N2A‐thin filament binding in mdm muscles reduces doublet potentiation. Using a servomotor force lever, we measured doublet potentiation in the soleus and extensor digitorum longus (EDL) muscles of wildtype and mdm mice at different muscle lengths. Potentiation was 20% lower in mdm than in wildtype soleus at all lengths (p = 0.04). In contrast to soleus, there was no difference in potentiation between wildtype and mdm EDL muscles (p = 0.7). In addition, potentiation was greater at shorter lengths than on the descending limb of the force‐length relationship in both soleus and EDL wildtype muscles. Mdm muscles did not show length dependence. Results from soleus muscles are consistent with the hypothesis that titin contributes to the increase in muscle force during doublet potentiation and that the lack of titin‐actin binding in mdm soleus muscles reduces doublet potentiation of muscle force. Differences in myosin isoforms, calcium flux, or titin isoforms, alone or in combination, may contribute to the observed differences between soleus and EDL in doublet potentiation.

Support or Funding Information

This work was funded by the Anderson Endowment at Denison University and NSF IOS‐1456868.
Peroxisomes in skeletal muscle protect against lipid‐induced insulin resistance
Authors: Noland Robert C. , Worsham Emily A. , Simon Jacob , Fuller Scott E. , Baes Myriam , Ghosh Sujoy , Mynatt Randall L.
Published: November 2023

Excess intramuscular lipids are thought to play a causal role in the pathogenesis of insulin resistance; thus, strategies aimed at reducing this lipid burden offer therapeutic potential. Peroxisomes provide an intriguing option to meet this goal as they have the ability to process a broad spectrum of lipid species. As such, the purpose of this study was to determine if skeletal muscle peroxisomes protect against lipid‐induced insulin resistance. To test this hypothesis, we developed a muscle‐specific peroxisome‐deficient mouse model by deleting Pex5 in skeletal muscle (Pex5m−/−) and weaned them onto a moderate fat (25%) diet to which they had ad libitum access for 20 weeks. Pex5m−/− mice had similar body weight and composition when compared to Pex5fl/fl littermate controls, but exhibited impaired glucose tolerance. This impairment in whole body glucose homeostasis was associated with a 25–30% decrease in mitochondrial function in Pex5m−/− mice, suggesting that peroxisomes helped protect against mitochondrial dysfunction in skeletal muscle. To further test this potential link between peroxisomes and mitochondria in skeletal muscle we analyzed peroxisomal adaptations in a murine model where mitochondrial fatty acid oxidation was limited via muscle‐specific deletion of the rate‐limiting enzyme of mitochondrial lipid entry, Cpt1b (Cpt1bm−/−). As expected, the limitation in mitochondrial fatty acid entry in Cpt1bm−/−mice resulted in a compensatory upregulation of peroxisomes which helped maintain adequate lipid oxidation. These adaptations were associated with robust improvements in glucose tolerance in Cpt1bm−/−mice. To test the importance of peroxisomal adaptations in mediating these improvements, we developed a Pex5:Cpt1bm−/−mouse model to prevent peroxisomal compensation in the Cpt1bm−/− strain. Results from these studies revealed that skeletal muscle fatty acid oxidation was severely compromised in Pex5:Cpt1bm−/−and that abrogation of peroxisomal function prevented the improvement in glucose tolerance that was incurred by muscle‐specific Cpt1b deletion alone. Overall these results suggest that peroxisomes in skeletal muscle play an important role in maintaining whole body glucose homeostasis and that this is likely at least partially attributed to the fact that peroxisomes provide an alternate route for lipid disposal which limits mitochondrial lipid overload.

Support or Funding Information

This work utilized PBRC core facilities (Genomics, Transgenic and Animal Phenotyping, and Animal Metabolism and Behavior) that are 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 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.