bluuebunny/crossref_metadata_2025 huggingface dataset:
TASK1 Channels as a pH Sensor in Mouse Adrenal Medullary cells: Analysis With Gene Knockout
Authors: INOUE Masumi , MATSUOKA Hidetada , HARADA Keita
Published: November 2023

Two‐pore K+ (2PK) channels constitute one of the K+ channel families, comprising 15 isoforms in humans. These isoforms, which are divided into six subfamilies, form a homodimer or heterodimer channel with isoforms in the same or different subfamilies. We have elucidated that acidosis induces catecholamine secretion through inhibiting TASK (TWIK‐related acid‐sensitive K+)‐like channels in rat adrenal medullary (AM) cells. TASK channels belong to the TASK subfamily in 2PK, comprising TASK1, TASK3, and TASK5. The expression of TASK1, but not TASK3, in rat AM cells was recognized by immunoblotting. However, RNAs for both TASK1 and TASK3 were clearly recognized with RT‐PCR in the rat adrenal medulla. Furthermore, in‐situ hybridization studies in mouse adrenal glands, strong and weak signals for TASK1 mRNAs were detected in adrenal cortex and adrenal medulla, respectively, and strong signals for TASK3 were recognized in both adrenal cortex and adrenal medulla. Thus, the present experiment was aimed to elucidate the molecular identity of TASK channels in mouse AM cells with the knockout (KO) of TASK genes. The perforated patch clamp technique was used to record the whole‐cell current. The inward current reversibly developed in response to step changes in external pH from 7.4 to 6.0 in wild‐type AM cells. The amplitudes of this acidosis‐induced current were well approximated by a logistic equation with IC50 of pH7.07. The pH‐dependent production of current was abolished by the genetic depletion of TASK1, but not TASK3. However, the holding current level at −50 mV and the slope conductance over the voltage range of −50 mV to −70 mV did not significantly differ between in wild‐type and TASK1‐KO AM cells. Immunocytochemistry revealed that TASK1‐like immunoreactivities (IRs) were mainly present at the cell periphery in wild‐type AM cells, but not in TASK1‐KO. TASK3‐like IRs were rarely detected in wild‐type AM cells and not enhanced in TASK1‐KO. A decrease in external solution induced catecholamine secretion in wild‐type AM cells, but not TASK1‐KO. These results indicate that TASK1 channels function as a pH sensor in mouse AM cells.

Support or Funding Information

Grants from Japan Society for the Promotion of Science and The Salt Science Research Foundation
Brain Perivascular Macrophages Contribute to Developing Hypertension via Sensing Chronic Peripheral Inflammation
Authors: Iyonaga Takeshi , Shinohara Keisuke , Kishi Takuya , Tsutsui Hiroyuki , Hirooka Yoshitaka
Published: November 2023

Background

Recent studies suggest an association between inflammation and hypertension. The essential pathophysiological mechanism by which peripheral inflammation induces sympathetic hyperactivity and blood pressure elevation is not fully clarified. Sympathetic nerve activity is regulated by the brain, which is separated from various peripheral substances by the blood brain barrier (BBB). Brain perivascular macrophages in the BBB can transfer peripheral inflammatory information to the brain. Therefore, we investigated whether peripheral proinflammatory cytokines accompany the development of hypertension, and if so, whether or not brain perivascular macrophage depletion attenuates the increase in blood pressure in hypertensive animal models.

Methods and Results

Peripheral proinflammatory interleukin‐6 levels measured by enzyme‐linked immunosorbent assay did not differ between young pre‐hypertensive spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY, 8‐week‐old SHR 64.8±9.5 pg/ml vs. 8‐week‐old WKY 42.8±9.5 pg/ml, p=0.15, respectively, n=4 for each). In adult hypertensive SHR, however, the interleukin‐6 level was significantly greater than that in WKY (12‐week‐old SHR 112.5±15.2 pg/ml vs. 12‐week‐old WKY 62.4±15.2 pg/ml, p<0.05, n=5 for each). Clodronate, a unique agent that induces apoptosis of perivascular macrophages, was administered intracerebroventricularly to 6‐week‐old SHR to investigate the role of brain perivascular macrophages in blood pressure elevation in SHR. Although established natural history of blood pressure in SHR clearly showed that mean arterial pressure would be elevated to approximately 120–125 mmHg at 8 weeks of age, intracerebrovantricular injection of clodronate successfully attenuated telemetered mean blood pressure elevation from 6 to 8 weeks of age (from 113.3±4.2 mmHg to 114.7±4.2 mmHg, n=2) in SHR.

Conclusion

Brain perivascular macrophages potentially contribute to developing hypertension via sensing chronic peripheral inflammation.
The Sex‐Specific Impact of Aging on the Blood Pressure Response to Exercise
Authors: Trinity Joel Douglas , Layec Gwenael , Hart Corey R , Richardson Russell S
Published: November 2023

An exaggerated increase in blood pressure (BP) during exercise has been linked to cardiovascular disease (CVD) and may precede the onset of overt CVD. Importantly, the prevalence of CVD is not evenly distributed between men and women across the lifespan and the impact of age and sex on the BP response to exercise remains unclear. Therefore, utilizing an integrative approach, this study examined central and peripheral hemodynamic responses and skeletal muscle metabolic responses to moderate intensity leg exercise in 40 activity‐matched young (n=20, 23±1 yr) and old (n=20, 73±2 yr) subjects. Central hemodynamics (finometer), popliteal artery blood flow (Doppler ultrasound), and skeletal muscle metabolic parameters (phosphorous magnetic resonance spectroscopy, 31P‐MRS) were measured during 5 min of submaximal dynamic plantar flexion exercise. Exaggerated exercise‐induced increases in systolic BP (SBP) and mean arterial pressure (MAP) were evident only in the old women (SBP: Δ47±6 mmHg, MAP: Δ30±4 mmHg) as all other groups exhibited similar increases in SBP (old men: Δ27±8; young men: Δ25±4; young women: Δ22±3 mmHg) and MAP (old men: Δ16±5; young men: Δ22±4; young women: Δ17±2 mmHg). Furthermore, unlike the young women, the old women failed to exhibit a reduction total peripheral resistance during exercise (young women: Δ −2.8±0.5, old women: Δ 1.0±1.0 mmHg/L/min, p < 0.05). Aging, independent of sex, was associated with attenuated central hemodynamic changes in stroke volume and cardiac output, while popliteal blood flow, O2 delivery, and high energy phosphate levels were similar between young and old. In conclusion, the combination of aging and the female sex results in an exaggerated BP response to exercise, likely a result of sustained elevations in total peripheral resistance during dynamic exercise.

Support or Funding Information

This project was supported by the American Heart Association (14SDG1850039) and the VA office of Research and Development (1IK2RX001215, E1433‐P, E1697‐R).
Housing temperature alters glucose metabolism in response to short‐term overfeeding in lean mice
Authors: Clayton Zachary Stephen , McCurdy Carrie
Published: November 2023

While diet induced obesity is an established risk factor for the development of insulin resistance, the exact mechanisms that initiate the insulin resistant phenotype are unknown. Systemic insulin resistance and glucose intolerance have been observed in as little as three days of high‐fat diet (HFD) feeding in lean mice. In most laboratories, mice are housed between 20–24°C, which is below their lower critical temperature (~30°C). Increased thermal stress has been shown to increase browning of white adipose tissue, alter lipid trafficking and impair immune function, which may lead to confounding results in metabolic data. Additionally, food intake and energy expenditure, two factors that significantly impact metabolic health, decrease with increasing ambient temperature. Thus, it is imperative to compare the metabolic and behavioral outcomes of high‐fat fed mice across typical housing temperature (22°C) and at their critical temperature (30°C). We hypothesized that mice will become glucose intolerant in as little as one day of HFD, and mice housed at 22°C will be protected from acute HFD induced impairments in glucose tolerance. To test this, we have studied an acute (1, 3 and 5 days) time‐course of either high‐fat feeding (45% Cal from fat) or low‐fat feeding (10% Cal from fat) in lean mice, at 22°C and 30°C. We observed a significant increase in glucose area under the curve (AUC) (P < 0.01) as soon as one day of HFD, which was not augmented by three or five days of HFD, without accompanied change in weight or body composition. However, separating our data by housing temperature, a significant increase in glucose AUC after one day of high‐fat feeding is only seen in mice housed at 30°C. Furthermore, the glucose AUC following one day of HFD at 30°C is similar to the glucose AUC observed after 5 days of HFD at 30°C. Behavioral outcomes such as food intake (significantly increased P < 0.01), oxygen consumption (significantly decreased (P < 0.05), and physical activity (significantly decreased P < 0.01) were also affected by one day of HFD relative to controls, and results were not exacerbated across three and five days. Taken together, we have shown that mice become glucose intolerant in as little as one day of HFD, and that the temperature at which the mice are housed is critical to accurately determining the metabolic phenotype.

Support or Funding Information

NIH DK095926‐01
Treatment with Streptozotocin (STZ) causes hypoglycemia and alters the stability of reference genes for real‐time PCR analysis in the liver of channel catfish
Authors: Nevarez Ericka , Ordonez‐Castillo Norelia , Spainhour Rebekah , Kobayashi Yass
Published: November 2023

Genetically selected channel catfish for increased growth often develops phenotype similar to human obesity, suggesting that channel catfish may be a model organism to investigate human metabolic disorders. Streptozotocin (STZ) treatment induces hyperglycemia in mammals and some fish species. However, the effects of STZ treatment on blood glucose level as well as other physiological parameters in channel catfish have never been examined in detail. In this study, we tested the hypothesis that treatment with STZ affects body weight, blood glucose level, and hepatic expression of house‐keeping genes. Juvenile channel catfish (28.3 ± 2g) were treated with 0, 3.6, 36, 180, or 360 mg/kg STZ intraperitoneally on day 0 (n=4 fish/treatment). On day 7, blood glucose level was measured and blood, liver, muscle and brain samples were collected. Expression of five common housekeeping genes (beta‐macroglobulin, alpha‐tubulin, 18s ribosomal RNA, beta‐actin, and GAPDH) were measured using real‐time PCR. The weight of fish did not change between days 0 and 7 among all treatments. Blood glucose levels were lower in fish treated with 180 or 360 mg/kg STZ compared to fish treated with 0 mg/kg STZ. Treatment with STZ at higher doses altered expression of 4 housekeeping genes. Expression of beta‐macroglobulin, alpha‐tubulin, and beta‐actin mRNA was higher in fish treated with higher doses of STZ compared to 0 mg/kg STZ. In contrast, expression of GAPDH mRNA tended to be less in 360 mg/kg STZ than 0 mg/kg STZ. Expression of 18s rRNA was not affected by STZ treatment. Rather than causing hyperglycemia, treatment with high doses of STZ decreased blood glucose level in channel catfish, suggesting a difference in glucose metabolism exists between channel catfish and humans.

Support or Funding Information

This study is supported by APS IOSP fellowship program and Kansas Idea Network of Biomedical Research Excellence (P20GM10348).
MicroRNA‐214–Mediated Suppression of Endothelial‐Notch2 Signaling Potentiates Vascular Endothelial Cell Migration and Proliferation in Pulmonary Arterial Hypertension
Authors: Sahoo Sanghamitra , Sembrat John C , Goncharova Elena A , Rojas Mauricio , Pagano Patrick J
Published: November 2023

Background

Pulmonary arterial hypertension (PAH) develops when pulmonary vascular resistance rises sharply, leading to right heart failure and death. Most studies have focused on attenuating lung vessel constriction; however, little is known about the mechanisms controlling intimal proliferation and luminal obstruction. Numerous studies suggest that the Notch signaling influences vascular cell proliferation and migration, hallmark features of neointimal growth. Despite this, a role for Notch2 in PAH is entirely unknown. We previously reported that microRNA‐214 (miR‐214) causes smooth muscle cell (SMC) proliferation and de‐differentiation. In this study, we hypothesize that miR‐214 inhibits lung endothelial Notch2 resulting in increased proliferation and migration in PAH.

Methods and Results

qPCR of human pulmonary artery endothelial cells (HPAECs, Lonza) showed that hypoxia (24 hr, 1% O2) upregulated miR‐214 by ~ 2‐fold. The miR‐214 rise was concomitant with reduction in Notch2 expression (0.61±0.08‐fold vs. normoxia, p< 0.05), and activation (0.50±0.01‐fold vs. normoxia, p< 0.01, by Western blot). Immunofluorescence staining on lung sections of PAH patients showed reduced expression of Notch2 in the intima of the vessel wall compared to non‐PAH samples. qPCR of pulmonary arteries from PAH patients showed significant reductions in Notch2 mRNA (0.30±0.08‐fold vs. control patients, p<0.05). Luciferase reporter assay demonstrated miR‐214 binding to Notch2 3′UTR with a marked reduction (~90%) in activity in HEK‐293 transfected with miR‐214 mimic compared to control. miR‐214 overexpression under normoxic conditions increased HPAEC migration (1.75±0.22‐fold vs. scrambled, p< 0.05), while hypoxia‐induced migration was attenuated by miR‐214 antagomiR. Finally, HPAEC pretreatment with Notch2 siRNA under normoxic conditions increased endothelial cell migration (1.5 ±0.01‐fold vs. scrambled control, p<0.05) and decreased the expression of p21cip (0.55±0.09‐fold vs. scrambled control, p<0.05).

Conclusions

The current study identifies a new signaling axis mediated by miR‐214‐Notch2 in the human pulmonary vascular endothelium that could contribute to pathophysiological changes to the intima and the progression of pulmonary arterial hypertension.

Support or Funding Information

This work was supported by National Institutes of Health grants, R01 HL112914 and P01HL103455 (PJP), R42‐AA024003 and R01‐CA162306 (MR) and R01HL113178 (EG). The study was supported by the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania.
Spirulina Extracts Improve Vascular Function of Arteries with Vascular Damage
Authors: Alameda M. Teresa , Ruiz Mireya , Villalpando Diva M. , Verdasco‐Martín Carlos M. , Plaza Ignacio , Otero Cristina , Ferrer Mercedes
Published: November 2023

Vascular dysfunction is characterized by an unbalance between relaxing and contracting factors, usually in favor of the latest ones, that maintained along the time could lead to cardiovascular diseases, such as hypertension. On the other hand, organ culture has been described to induce phenotypic changes in endothelial cells similar to those observed in some cardiovascular pathologies. In both hypertension and in organ culture‐induced vascular damage, the release and action of nitric oxide (NO) play a pivotal role in the control of vascular function. Recently, increasing attention is paying to the use of the microalgae Spirulina for nutritional interventions due to antioxidant and anti‐inflammatory properties. Therefore, the aim of this study was to analyze the effects of aqueous Spirulina extract on the release and function of NO in these two experimental models of vascular dysfunction. For this purpose, thoracic aorta from Sprague‐Dawley (SD), spontaneously hypertensive rats (SHR) and its normotensive Wistar‐Kyoto (WKY) rats (18–20 weeks old) were used. Organ culture‐induced vascular damage was obtained by incubating aortic segments from SD rats for 20 hours at 37° C in serum free culture medium, and the effect of aqueous Spirulina extract (0.1 % w/v; for 3 h) on the NO release and on the vasodilation induced by acetylcholine (Ach) were analyzed. In aortic segments from SHR and WKY rats, the effect of Spirulina extract on NO release and on the Ach‐induced response in endothelium‐intact and endothelium‐denuded arteries were investigated. The results showed that the induced vascular damage strongly increased the release of NO, while the incubation with the Spirulina extract decreased that release up to similar levels found in intact arteries. The Ach‐induced response was not modified after vascular damage or Spirulina incubation. In aortic segments from SHR and WKY rats Spirulina extracts increased the release of NO more in arteries from SHR than that of WKY rats. In endothelium‐intact arteries, Spirulina extracts increased more the Ach‐induced response in WKY rats than in those of SHR. Conversely, in endothelium‐denuded arteries, Spirulina increased the Ach‐induced relaxation more in SHR than in those of WKY rats. These findings indicate the beneficial role of Spirulina extracts on the NO participation in the vasodilator responses of these two experimental models of vascular dysfunction, although endothelium‐independent mechanisms are also involved.

M.T.A. and M.R. equally contributed to this work

Support or Funding Information

Supported by grants (to M. F.) from the Fondo de Investigaciones Sanitarias (PI1100416), Comunidad de Madrid (S2013/ABI‐2783, “INSPIRA1‐CM”) and Fondo Europeo de Desarrollo Regional.
Energy Homeostasis Is Disrupted During Recovery From Heat Stroke
Authors: Mayer Thomas A , Leon Lisa R , Audet Gerald N
Published: November 2023

Heat stroke is a life‐threatening medical condition that affects thousands of Service Members and civilians every year. Loss of function of various physiological processes occurs during recovery as multisystem organ dysfunction/failure ensues. Splanchnic organs, which influence energy homeostasis, are at a particularly high risk of damage due to ischemia/reperfusion injury during and after heat stress. This study sought to determine whether energy homeostasis was disrupted during heat stroke recovery. Conscious adult male Fisher 344 rats previously implanted with a radiotelemetry device to measure physiological vitals (e.g. temperature, heart rate) were heated until core temperature (Tc) reached 42.0±0.1°C; control rats were not heated. Heat stroke severity was categorized as mild, moderate, or severe based on physiological responses and organ histopathology. Rats were sacrificed one day after heat exposure and tissues and plasma were collected for analysis. Severe and moderate (mod) rats exhibited lower blood glucose levels than controls (ctrl) (ctrl 189.3±2.8; mod 154.0±6.4; severe 148.8±10.5 mg/dL; p<0.05). Glycogen stored in the liver was reduced in heated rats compared to controls and further reduced in moderate and severe rats compared to mild rats (ctrl 50.4±2.3; mild 28.3±4.8; mod 11.2±4.3; severe 6.6±4.2 mg/g liver tissue; p<0.05). In plasma, the primary ketone body beta‐hydroxybutyrate was elevated only in severe rats (ctrl 0.24±0.01; mild 0.22±0.02; mod 0.18±0.02; severe 0.47±0.08 nmol/μL; p<0.05). Circulating albumin was reduced in heated rats compared to controls and further reduced in severe rats compared to mild rats (ctrl 4.66±0.06; mild 4.11±0.11; mod 3.83±0.14; severe 3.46±0.12 g/dL; p<0.05). Non‐fasted insulin levels trended lower in moderate and severe rats compared to controls. These data support previous findings that heat stroke results in a diminished capacity to regulate glucose homeostasis, which appears to worsen with severity. Decreased albumin levels found in this study may suggest hindered fatty acid mobilization. In conclusion, energy reserves in heat stroke patients should be monitored to promote recovery. Author views not official US Army or DoD policy.
Dynamic resistance training improves cardiovascular autonomic variability in male hypertensive rats
Authors: Araujo Amanda , Dias Danielle , Bernardes Nathalia , Conti Filipe , Allioni Gabriela , Irigoyen Maria Cláudia , De Angelis Kátia
Published: November 2023

The dynamic resistance training (DRT) has been the focus of recent researches; however, few studies evaluated the effects of DRT on hemodynamics of hypertensive subjects. The objective of this study was to evaluate the effects of DRT on blood pressure (BP) and cardiovascular autonomic modulation in spontaneously hypertensive rats. Thus, we used 16 adult males SHR, divided into two groups (n = 8/group): sedentary (SM) and trained (TM). Maximal exercise testing was conducted before and after the training period. The DRT, in moderate intensity (40–60% of the maximal load), was performed in a ladder adapted for rats per 8 weeks. After this period, blood glucose and triglyceride were measured and catheter was inserted in the carotid artery for direct measurement of BP (WINDAQ, 2 KHz). BP and heart rate variability were evaluated in time and frequency domains. At the end of the protocol, there was an improvement in physical capacity in the TM group (TM: 737±39 vs. SM: 476±50 g). Moreover, there were no differences in the weight of left ventricle and soleus muscle; however, the TM group showed an increase in plantar muscle (0.26±0.01 g) when compared to SM group (0.20±0.02 g). There was no difference in heart rate (TM: 350±8 vs. SM: 366±10 bpm) and blood glucose (TM: 88±3 vs. SM: 85±2 mg/dl); however, TM group showed reduced blood triglycerides values (TM: 110±7 vs. SM: 133±4 mg/dl). DTR did not induced reduction in mean, systolic and diastolic BP (mean BP, TM: 186±4 vs. TS: 185±9 mmHg). Despite that, DRT improved cardiac vagal modulation demonstrated by the increase in SD‐IP (TM: 6.61±1.31 vs. SM: 3.68±0.32 ms) and RMSSD (TM: 3.95±0.30 vs. SM: 3.02±0.25 ms). A reduction in BP variance (TM: 33±3 vs. SM: 66±4 mmHg2) and vascular sympathetic modulation (LF‐BP, TM: 6±2 vs. SM: 12±1 mmHg2) were observed in trained group. Additionally, DRT induced an improvement on spontaneous baroreflex (TM: 0.39±0.04 vs. SM: 0.22±0.01 ms/mmHg). We conclude that moderate intensity DRT in male SHR unchanged resting BP, but induced improvement on cardiovascular autonomic variability. These results suggesting a positive role of this approach, associated with aerobic exercise training, on hypertension management.

Support or Funding Information

FAPESP 2015/10329‐5, 2015/11223‐6, UNINOVE.
Dystrophin Insufficiency Increases Skeletal Muscle Damage
Authors: Hill Sydney , Lien Stuart , Spaulding Hannah , Nonneman Dan , Ross Jason W , Selsby Joshua T
Published: November 2023

Dystrophinopathies are caused by a mutation in the dystrophin gene, which can result in dystrophin protein insufficiency (Becker muscular dystrophy) and more commonly a dystrophin protein deficiency (Duchenne muscular dystrophy). These diseases are frequently modeled by the mdx mouse or the Golden Retriever Muscular Dystrophy model. Albeit useful, these models are not optimal for translational research due to inherent limitations. The purpose of this research was to determine the extent to which skeletal muscle is damaged by dystrophin insufficiency in a spontaneously occurring pig muscular dystrophy model. We hypothesized that dystrophin insufficiency would result in progressive muscle injury to skeletal muscle. To test this hypothesis, diaphragm from male pigs affected by dystrophin insufficiency and healthy male littermates were harvested at 3 months and 12 months of age and frozen for subsequent histological analysis. Sections were stained with hematoxylin and eosin and 3–5 random images were taken of each section. Central nucleation, a marker of skeletal muscle degeneration/regeneration, was similar between groups at 3 months of age, however, at 12 months of age central nucleation was increased by 42% in affected pigs compared to healthy pigs. When considering changes over time central nucleation increased in healthy pigs by 5‐fold (3 mo – 1.5%; 12 mo – 8%; p<0.05) and by 8‐fold in affected pigs (3 mo – 1.8%; 12 mo – 14%; p<0.05). These data indicate that the porcine diaphragm undergoes an increasing rate of central nucleation with normal aging. Importantly, this rate is dramatically increased with dystrophin insufficiency suggestive of an elevated pathological degeneration and regeneration. Interestingly, extracellular nuclei, which are associated with immune cell infiltration, and total muscle cells per area changed as a function of time but were similar between healthy and affected pigs at both 3 and 12 months of age. These data suggest that dystrophin insufficiency in diaphragm muscle increased muscle damage, which is consistent with disease‐related injury.

Support or Funding Information

Supported by NIH R21 NS079603
Growth Arrest Specific 6 Activates Akt and ERK in Cultured Skeletal Muscle
Authors: Olsen Esther K. , Doxey Stephen A. , Chow Joshua L.Y. , Duplisea Michael , Thomson David M.
Published: November 2023

Growth arrest specific 6 (Gas6) is a signaling factor that is found in a variety of tissues and is thought to be involved in the regulation of cell proliferation through activation of AKT and/or ERK signaling pathways. Its effect on skeletal muscle cells is unknown. Therefore, to test if Gas6 activates AKT and ERK in skeletal muscle, we incubated C2C12 myoblasts and myotubes with 0 to 800 ng/ml of Gas6 for 15 minutes. The cells were lysed and AKT and ERK content and phosphorylation were measured by western blotting. We found that in myoblasts Gas6 significantly increased ERK, but not AKT phosphorylation at an optimal concentration of 100 ng/ml. In myotubes, Gas6 significantly increased AKT but not ERK phosphorylation, also at an optimal concentration of 100 ng/ml. Our findings indicate that Gas6 is able to regulate these pathways in skeletal muscle and/or skeletal muscle progenitor cells. This suggests that Gas6 may be useful in conditions where muscle growth and/or satellite cell proliferation are impaired such as diabetes and old age.

Support or Funding Information

This work was supported by a Brigham Young University Gerontology Program Grant.
A single bout of cryotherapy does not alter the transcriptome or metabolome of human skeletal muscle
Authors: Sarver Dylan C , Sugg Kristoffer B , Disser Nathaniel P , Enselman Elizabeth R Siblinsky , Mendias Christopher L
Published: November 2023

Cryotherapy is a commonly used therapeutic modality for skeletal muscle injuries in musculoskeletal medicine. Despite the widespread use of this modality, there is little known about the biochemical effects of cryotherapy in human skeletal muscle tissue. The purpose of this study was to determine the effects of a clinically relevant dose of cryotherapy on the transcriptome and metabolome of muscle tissue, as well as the activation of various signaling proteins. Using a paired design, 8 healthy male subjects (mean age 24.7±4.5 years, mean BMI 22.2±1.6) received ice‐cup massage or sham cup massage over a 45 cm2 area on each thigh for 15 min. Two hours after application, bilateral biopsies were taken from the vastus lateralis at a depth of 2 cm in the center of the ice or sham area. Muscle biopsies from each leg were then subjected to microarray, LC/MS‐based metabolomics analysis, or Western blot. Differences between groups were tested using paired t‐tests (α=0.05). Intramuscular (IM) temperature 2 cm deep to the subcutaneous layer was predicted from regression equations of skin temperature. Prior to administration of cryotherapy, intramuscular (IM) temperature was predicted to be 35.0±0.5°C. At the end of the 15 min application, IM temperature was reduced to 25.2±1.4°C (P<0.001), and by two hours remained below baseline at 31.7±0.8°C (P<0.001). Microarray analysis revealed changes in some non‐coding RNAs, but no differences were found for protein coding genes. Further analysis by qPCR showed no significant differences in cold‐shock genes which have been reported to be induced in animal tissue exposed to extended cooling. Metabolomics analysis of over 60 metabolites involved in glycolysis, oxidative phosphorylation, and amino acid metabolism showed significant (p<0.05) decreases in the hexose sugars and hypoxanthine by 15% and 17%, respectively, in cooled skeletal muscle tissue compared to controls. No differences in other metabolites were detected. Western blot analysis of major proteins involved in protein synthesis (p‐Akt, p‐mTOR, p‐p70S6K) showed no differences between groups. In summary, a clinically relevant administration of cryotherapy does not seem to have a significant, acute impact on the transcriptome, metabolome, or protein synthesis pathways in otherwise healthy muscle tissue.

Support or Funding Information

NIH grants F32‐AR067086 and U24‐DK097153
Study of Acute Kidney Injury in hyperandrogenemic female rats
Authors: Patil Chetan N , Dalmasso Carolina , Maranon Rodrigo O. , Yanes Cardozo Licy L. , Reckelhoff Jane F.
Published: November 2023

Chronic hyperandrogenemia is hallmark of polycystic ovarian syndrome (PCOS), affecting ~10% of women of reproductive age. Compared to men, women are protected against ischemia‐ reperfusion (I/R) acute kidney injury (AKI), but the potential susceptibility of women that have elevated androgens, as women with PCOS, to develop I/R‐induced AKI is not clear. Other models of hyperandrogenemia in females in which I/R AKI was studied utilized levels of testosterone similar to males. Using an established female rat model of PCOS that has a 3 fold increase in DHT, such as women with PCOS and 10 fold lower than in males, we tested the hypothesis that chronic hyperandrogenemia increases the risk of I/R‐induced AKI compare to placebo controls. Female SD rats were implanted with dihydrotestosterone (DHT, 7.5mg/90d) or placebo pellets (n=11/grp) beginning at 6 wks of age; pellets were changed every 85 d. At 6–7 mos of age, rats (n=3–4/grp) were subjected to sham or renal I/R with bilateral clamping of renal vessels for 30 min at 37±0.5°C. Rats were placed in metabolism cages for 24hrs reperfusion. At 48–72hr post‐surgery rats were again put in metabolic cages for 24hr, and then euthanized for blood and tissue samples. I/R increased plasma creatinine (PCr) 24hr post reperfusion in placebo (2.58±0.34 vs 0.86±0.1 mg/dL, p<0.05) as well as DHT treated rats (4.53± 0.46 vs 0.9±0.06 mg/dL, P<0.001) compared to sham‐I/R group. As noted, PCr in DHT‐treated rats was significantly higher than in placebo rats (4.53± 0.46 vs 2.58±0.34 mg/dL, p<0.05). When DHT rats were pre‐treated with androgen receptor antagonist (Flutamide, 30mg/kg/day, s.c.) for three days prior to I/R, PCr levels 24hr post reperfusion were not different than DHT–treated I/R groups (4.75 0.52 vs 4.53± 0.46 mg/dL, p=ns). These data show that rat exposure to chronic hyperandrogenemia causes significantly greater renal injury in response to I/R AKI than in controls. This injury is independent of the androgen receptor suggesting other mechanisms may be contributing to the higher level of renal injury after I/R AKI in females with PCOS.

Support or Funding Information

Supported by NIH R01HL66072, P01HL05971.
Combined hydroxyurea and ETA receptor blockade on renal dysfunction in the humanized sickle mouse
Authors: Taylor Crystal Monique , Kasztan Malgorzata , Tao Binli , Pollock Jennifer S , Pollock David
Published: November 2023

Sickle cell disease (SCD) is associated with an increasing incidence of kidney disease, and the underlying causes have not been elucidated. Recent data has from our lab and others have shown that the vaso‐active peptide endothelin‐1 (ET‐1) plays a critical role in the pathophysiology of sickle cell nephropathy (SCN) and the blockade of its ETA receptor improves renal function and reduces renal injury. As hydroxyurea (HU) is a commonly prescribed drug for the treatment of SCD due to its ability to increase fetal hemoglobin, investigating how these two complex systems interact in the pathophysiology of SCN could provide insight into pathophysiologic mechanisms. Therefore, we hypothesized that combined ambrisentan (ETA selective antagonist) and HU treatment has a synergistic effect on renal injury in SCN when compared to HU treatment alone. Male 12 week old humanized sickle mice (Towne's mice; HbSS) and their genetic controls (HbAA) were treated with either vehicle (drinking water), HU (50mg/kg/day), or HU plus ambrisentan (10mg/kg/day). After 2 weeks of treatment, mice were placed in metabolic cages to assess renal function and then euthanized for blood and tissue collection. Vehicle treated HbSS mice exhibited significant proteinuria (4.38 ± 0.89 mg/day, p=0.0082), elevated plasma ET‐1 concentration (2.68 ± 0.44 pg/ml, p=0.01), and urinary ET‐1 excretion (1.43 ± 0.17pg/day, p=0.0042) as well as decreased urine osmolarity (1205 ± 63.3 mOsmol/kg, p<0.0001) compared to HbAA controls. This renal injury phenotype was significantly attenuated in the HU treated animals (2.09 ± 0.28 mg/day, 0.881± 0.100 pg/ml, 0.336 ± 0.05 pg/day, 1686 ± 83.1mOsmol/kg, respectively; p<0.05 compared to HbSS vehicle treated for all analyses); however, there was no additional improvement in HbSS mice treated with combined ambrisentan and HU. The absence of further attenuation of renal injury with combined treatment suggests that the mechanism of action for both treatments may converge on the same mechanistic pathway.
High Protein Diets Blunt Insulin‐Stimulated Akt Phosphorylation While Enhancing Fibroblast Growth Factor Receptor Phosphorylation in Rat Liver
Authors: Noble Nicole D , Pettit‐Mee Ryan J , Cunningham Rory P , Sharma Naveen
Published: November 2023

The activation of the fibroblast growth factor (FGF) pathway in the liver has been demonstrated to regulate hepatic metabolism of lipids, cholesterol, and bile acids. Irregularities in liver metabolism can result in many different metabolic disorders, which can be treated with various dietary interventions. However, the impact of high protein diets on FGF signaling in the liver remains unclear. Therefore the purpose of this study was to investigate the effect of a high casein‐based or a high soy‐based protein diet on signaling proteins associated with the FGF pathway within hepatic tissue. For 15 weeks, Sprague‐Dawley rats were fed either a control diet (CON; consisting of 20% casein protein by calories), a high casein protein diet (CAS; 40% casein protein), or a high soy protein diet (SOY; 40% soy protein). A terminal surgery was performed after an overnight fast. Under isoflurane anesthesia, saline or insulin (10U/kg) IP injections were administered to the rodents, then after 5 minutes, blood and liver samples were collected. Plasma levels of insulin and FGF‐21 were determined by ELISA. Tissue levels of FGF‐21, phosphorylated FGF receptor (pFGFR1Tyr654), and phosphorylated Akt (pAktSer473) were determined by Western immunoblot. A two‐way ANOVA was used to determine main effects of diet and insulin on plasma and tissue analytes. As expected, insulin injection resulted in a significant (P<0.05) increase in plasma insulin levels and increased pAktSer473 in the liver tissue compared to saline‐injected samples. Additionally the insulin‐induced elevation of pAktSer473 in the liver was blunted in the CAS and SOY groups compared to the CON group. There was a main effect of insulin for pFGFR1Tyr654 in the liver (insulin > saline; P<0.05). There was a main effect of diet for FGF‐21 in the liver (CAS < CON and SOY; P<0.05) while conversely plasma levels of FGF‐21 were significantly (P<0.05) elevated in plasma of the CAS group compared to either CON or SOY groups. Our findings reveal that high protein diets blunt the insulin‐enhanced pAktSer473. We speculate that high protein diets may enhance insulin‐stimulated FGF signaling in the liver independent of Akt‐mediated pathways. The therapeutic implication of this observation is yet to be determined.

Support or Funding Information

Central Michigan University Herbert H. and Grace A. Dow College of Health Professions, and the Office of Research and Graduate Studies Early Career Grant.
Stereological Analysis Of Long‐Term Effect Of Very High Fat Diet To Study The Synergism In Hormonal And Cellular Changes In Male Mice
Authors: Chakraborty Tandra Roy , Gomez Victoria , Babu Sathyapriya , Gurung Devya , Caraballo Trevor , Chakraborty Sanjoy
Published: November 2023

Objective

To determine the hormonal changes that occur as a result of the long term intake of a very high fat diet (VHFD) leads to simultaneous changes in the islets of Langerhans and adipocyte cell size.

Methods

Male mice were fed with a normal chow diet (ND, n=15) and a VHFD (n=30) for 2, 12 and 24 weeks. Body weight, food intake, caloric intake [fat (saturated and unsaturated), protein and carbohydrate], hormone levels (leptin and insulin), and islet of Langerhans/adipocyte size were quantitatively recorded using stereology.

Results

In VHFD‐fed animals, body weight showed significant percent increase within the first 12 weeks and then plateaued with time. VHFD‐fed animals consumed significantly less food than ND at all time periods indicating that it was the quality of food and not the quantity that caused this increase in body weight. Stereological analysis of male mice on VHFD showed significant increase in leptin and insulin levels along with accompanying growth in islet and adipocyte size within the first 12 weeks which plateaued as the mice aged. The increases in the islet and adipocyte size in VHFD fed animals were similar to the analogous increases in hormonal levels (2 vs 12 vs 24 weeks). These results, therefore, suggest that in diet‐ induced obesity changes, shifts in hormonal levels works hand in hand with metabolic adjustments at the cellular level to combat the effect of fat.

Conclusion

Thus, hormonal resistance, changes in adiposity, islet size and caloric intake with prolonged exposure to high fat are probably defensive mechanisms employed to protect against diabetes. In order to understand these complicated and nuanced effects of high fat and to comprehend the underlying mechanism, it is important to focus on long‐term studies that emphasize the synergy between cellular and hormonal changes in addition to an analysis of individual components.
Effects of a TRPV4 Antagonist on Hydrocephalus in Wpk Rat Model
Authors: Danko Caleb , Preston Daniel , Simpson Stefanie , Blazer‐Yost Bonnie
Published: November 2023

Hydrocephalus is a disease classified by an excess of Cerebrospinal Fluid (CSF) in the brain resulting from a variety of causes. Current treatments for hydrocephalus are limited to invasive surgical procedures that have high risks of failure or infection. The Choroid Plexus (CP) is responsible for production of the CSF, and is found in the lateral, third, and fourth ventricles of the brain. The CP contains a high resistance epithelium that tightly regulates the ionic and water movement during the production of CSF. The Wpk rat model has a single point mutation that induces a ciliopathy in homozygous recessive allele expression leading to hydrocephalus and polycystic kidney disease. The hydrocephalus in the Wpk rat model is a homologue to pediatric hydrocephalus that mimics a rapid progression of the disease, resulting in death of the rats, without intervention, within the first 21 days of life. Considering the tight control of fluid/electrolyte regulation in the CP, we have explored an osmotic‐ and mechano‐sensitive Ca2+ influx channel the transient receptor potential vanilloid‐4 (TRPV4), as a potential target for pharmaceutical regulation of CSF production. The Wpk rat pups are given a daily, intraperitoneal injection of the TRPV4 antagonist, RN1734 starting on day 7 after birth (P7), continuing through day 14 (P14), with untreated animals receiving a vehicle injection. Body weight is recorded daily with injections calculated for controlled drug doses according to body weight. Wpk rats were humanely euthanized on P15 with measurements taken of the body weight, kidney weight, cranial dimensions, and brain weight. These data were expressed in several parameters to account for the differences in body weight between the hydrocephalic and wild type pups as well as to account for the increase in cystic kidney weight in homozygous animals. Drug treatment had no effect on body weight, head dimensions or behavior of the wild‐type animals. However, in the hydrocephalic animals, head size in both the vertical and horizontal dimension were statistically decreased by antagonist treatment. These relationships were valid even when body weight or anephric body weights were taken into consideration. Likewise the brain weights were statistically lowered by treatment in the hydrocephalic animals. In this genetic model of hydrocephalus, treatment with a TRPV4 antagonist substantially ameliorates the hydrocephalic development suggesting that this class of drugs might be a good target for pharmaceutical development.

Support or Funding Information

Funding: Hydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Influence of Intrathoracic Vagotomy On the Cough Reflex in the Anesthetized Cat
Authors: Bolser Donald C , Shen Tabitha Y , Rose Melanie J , Musselwhite M Nicholas , Davenport Paul W
Published: November 2023

Cervical vagotomy sections both lower airway and recurrent laryngeal afferent fibers and eliminates cough induced by stimulation of sensory afferents in the lower trachea and intrathoracic airways. In the guinea pig, recurrent laryngeal afferent fibers are primarily responsible for cough in response to mechanical or chemical stimulation of the upper trachea and larynx. Further, it has been proposed that lower airway slowly adapting receptors have a permissive effect on the cough reflex. The objective of this study was to determine the role of lower airway vagal afferent fibers on the cough reflex in the cat. We hypothesized that cough induced by mechanical or chemical stimuli applied to the lower airways or the larynx of this species would be depressed after vagotomy below the branch of the recurrent laryngeal nerve. Experiments were conducted in four anesthetized, bilaterally thoracotomized, and artificially ventilated cats. Artificial ventilation was accomplished via a tracheal cannula placed through an incision in the tenth trachea ring. Mechanical (via a flexible cannula through a small port in the trachea tube) or chemical (capsaicin, 0.5 cc, 10 μM) stimulation of the intrathoracic airway was employed during mechanical ventilation. Thoracic vagotomy virtually eliminated the cough response to mechanical stimulation of the lower airways (control cough number 4.7±1.7; after vagotomy 0.2±0.2 coughs, P<0.04). The cough response to mechanical stimulation of the larynx was also depressed after thoracic vagotomy. Capsaicin introduced into the intrathoracic trachea only elicited cough in two animals but this response was eliminated in both animals by thoracic vagotomy. The results support an important role of lower airway sensory feedback in the production of cough in the anesthetized cat. Further, in the presence of intact recurrent laryngeal nerves, the excitability of cough induced from the larynx is strongly influenced by sensory feedback from the lower airways.

Support or Funding Information

NIH 1OT20D001983
Epigenetic modifications influence TLR3 expression and activity
Authors: McKernan Declan Patrick , Hennessy Conor
Published: November 2023

Background

Toll‐like receptors (TLRs) are a family of pattern recognition receptors (PRRs) which recognise conserved pathogen associated molecular patterns (PAMPs) present in bacteria and viruses e.g. TLR3 recognises dsRNA from viruses. TLRs are expressed on a variety of cells including intestinal epithelial cells and play an important role in the immune response by stimulating cytokine release via phosphorylation of signalling proteins, but it is not known how their expression is regulated. We hypothesised that epigenetic mechanisms such as DNA methylation and histone deacetylation may play a role. In order to investigate this, we studied the effect of inhibition and/or genetic knockout of the enzymes responsible for these events (DNA methyltransferases (DNMT) or histone deacetylase complexes (HDAC)) on TLR3 expression and signalling in HCT‐116 intestinal epithelial cells.

Methods

HCT‐116 (both wild‐type (WT) and DNMT 1/3B double knockout (DKO) cells) cells were either untreated or treated with the pan‐HDAC inhibitor suberanilohydroxamic acid (SAHA, 10μM, 48hrs), or the DNMT inhibitor 5‐aza‐2‐deoxycytidine (500nM, 72hrs), followed by stimulation with the TLR3 ligand Poly I:C (10μg/ml, 6 or 24 hrs). Cytokine mRNA expression was measured using quantitative polymerase chain reaction (QPCR) while protein release was measured using ELISA of the cell culture supernatant. Intracellular signalling protein phosphorylation was measured using Western blotting. Statistical analyses was performed in SPSS and in all cases n ≥ 3.

Results

Treatment of HCT‐116 WT cells with Poly I:C resulted in a significant increase in TLR3 expression (p<0.001) and in the mRNA expression of cytokines (p<0.001) and also the release of cytokines (p<0.001). Poly I:C treatment also resulted in increased phosphorylation of signalling proteins (NF‐κB, IκBα, JNK, p38, IRF3). Pre‐treatment with SAHA, 5‐aza‐2‐deoxycytidine and genetic knockout of DNTM1/3b prevented this Poly I:C induced increase in TLR3 expression and cytokine mRNA expression, cytokine release and signalling protein phosphorylation.

Conclusion

Inhibition of HDACs or DNMTs by pharmacological means or genetic knockout prevents TLR3 signalling and the activation of the TLR3 inflammatory pathway. This suggests that viral induced inflammation may be controlled using epigenetic modifying drugs.

Support or Funding Information

College of Science Scholarship, NUI Galway