bluuebunny/crossref_metadata_2025 huggingface dataset:
The α7β1 Integrin Directly Regulates SLC7A5 (LAT1) Amino Acid Transporter Transcription in Skeletal Muscle
Authors: Mahmassani Ziad , Son Kook , Chen Jie , Boppart Marni
Published: November 2023

INTRODUCTION

The α7β1 integrin is a focal adhesion protein found at the costameres of skeletal muscle and it provides a critical link between the actin cytoskeleton and laminin in the basement membrane. We have demonstrated that overexpression of the α7BX2 integrin subunit in muscle (MCK:α7BX2) enhances myofiber growth in the gastrocnemius following multiple bouts of eccentric exercise. Results from a recent microarray analysis suggest that the α7β1 integrin alone, in the absence of mechanical strain, initiates transcription of genes that allow for protection from stress, modulation of protein synthesis and regulation of substrate utilization.

PURPOSE

The purpose of this study was to determine the extent to which the α7 integrin can directly regulate transcription of targeted genes using in vitro analysis.

METHODS

Two separate small hairpin RNAs (shRNAs) in the pLKO.1‐puro vector for mouse ITGA7 were virally transduced into C2C12 myoblasts, with a scramble vector as a control. Myoblasts were differentiated and targeted gene expression (RPL13a, NOSIP, ANG, SLC7A5, GYS1, NDRG2, HSP40, HSPa5, and ATF6) was assessed using qPCR.

RESULTS

shRNA knockdown of the α7 integrin using either shRNA construct in C2C12 myotubes suppressed gene expression of the amino acid transporter SLC7A5 (LAT1), yet one increased and the other suppressed NDRG2, a stress‐responsive regulator of protein synthesis in striated muscle (P < 0.05).

CONCLUSION

The results from these studies suggest that the α7β1 integrin can directly regulate transcription of both SLC7A5 and NDRG2 in skeletal muscle under baseline (non‐exercised or loaded) conditions. The majority of alterations in gene transcription observed with array analysis, however, likely occur in cells outside the myofiber. Future studies are necessary to determine if integrin expression provides the basis for altered proteostasis in the context of disuse and disease.

Support or Funding Information

NIH NIAMS 1 R21 AR065578A, NIH NIAMS R01AR048914.
Ribosome biogenesis is activated during the early period of resistance training in rat skeletal muscle
Authors: Kotani Takaya , Takegaki Junya , Nakada Satoshi , Takagi Ryo , Tomiya Shigeto , Tsutaki Arata , Nakazato Koichi , Ishii Naokata
Published: November 2023

Resistance training (RT) induces skeletal muscle hypertrophy. Recently, it has been suggested that ribosome biogenesis plays an important part in RT‐induced skeletal muscle hypertrophy. However, influence of number of RT bouts is unclear.

Purpose

The present study aimed to investigate the change of ribosomal RNA (rRNA) and ribosome protein content with changing the number of RT bouts.

Method

Male Sprague‐Dawley rats were randomly assigned into six groups: Control (Cont), resistance‐trained with 1bout (1B), 2bouts (2B), 3bouts (3B), 6bouts (6B) and 18bouts(18B). RT protocol consisted of 50 repetitions of maximal isometric contraction of the gastrocnemius muscle by direct electric stimulation. Groups other than 1B performed 3 bouts of training per week. Muscle samples were collected 3h, 6h and 48h after the final exercise session in 1B to 3B, and 48h after the final exercise session in 6B and 18B.

Results

The content of 18S+28S rRNA and rpS6 increased in 1B to 3B, but no significant difference was seen between 3B, 6B and 18B.

Conclusion

The present showed that ribosome biogenesis is activated during the early period of RT. These results suggested that activation of ribosome biogenesis may contribute to increase protein synthesis during the later period, thereby leading to muscle hypertrophy.
Two Weeks of Reduced Steps Disrupts Integrated Rates of Muscle Protein Synthesis and Glycemic Control in Healthy Older Adults
Authors: McGlory Chris , Stokes Tanner , von Allmen Mark , Morton Robert , Smith Brennan , Steinberg Gregory , Phillips Stuart
Published: November 2023

Transient periods of physical inactivity can result in losses of skeletal muscle mass and reductions in insulin sensitivity. These metabolic impairments may not be fully recovered, particularly in older individuals.1 We aimed to examine the impact of two weeks of reduced steps followed by two weeks of recovery, on lean body mass (LBM), integrated rates of myofibrillar protein synthesis (MPS), single fiber cross‐sectional area (fCSA), and indices of glycemic control (HOMA‐IR and MATSUDA) in response to an oral glucose tolerance test. We also examined changes in oxidative phosphorylation protein complex content, phosphorylation of other related signaling molecules, and concentration of circulating inflammatory markers. Twenty‐two healthy older adults (12 men, 10 women, 69±4yr [all data mean±SD]) underwent 7d of monitored normal activity at baseline (BL) before reducing their daily step‐count from 7362±3294 to 990±97 steps.d−1 for 14d (SR). Habitual step count was then recovered for a period of 14d (RC). Integrated rates of muscle protein synthesis (MPS) were reduced from BL at SR (BL 1.51±0.07 to SR 1.33±0.05 %.d−1, p<0.05) and were not restored at RC (1.34±0.14 %.d−1). Women displayed significantly greater rates of integrated MPS at BL compared to men (1.56±0.06 vs. 1.47±0.04 %.d−1, p<0.05). HOMA‐IR increased from BL to SR (2.6±0.4 to 3.8±0.9, p<0.05) and MATSUDA decreased (3.9±0.7 to 2.9±0.4, p<0.05), neither recovered at RC. Glucose AUC was significantly elevated from BL at SR (368±137 to 442±142, p<0.05) and was not fully recovered from SR at RC (393±15). Insulin AUC increased from BL to SR (3372±889 to 3972±1008, p<0.05) and was still elevated at RC (3865±716). Circulating plasma TNF‐a was significantly increased from BL at SR (11.78±2.84 to 15.41±4.64 pg.mL−1, p<0.05) and was still elevated above BL at RC (14.28±3.76 pg.mL−1). Plasma IL‐6 concentration was also significantly increased from BL at SR (8.33±3.06 to 10.84± 4.35 pg.mL−1, p<0.05) and remained elevated at RC (9.99±4.37 pg.mL−1). There was no detectable impact of step reduction on LBM, fCSA, fiber type distribution, or the content of oxidative phosphorylation protein complexes at any time point. Additionally, step reduction failed to impact the phosphorylation status of ACCSer212, AktSer473, mTORSer2448, Ulk1Ser757, and Ulk1Ser55 at BL, SR, and RC. These data demonstrate that two weeks of step reduction leads to lowered rates of MPS that are accompanied by appearance of a diabetic phenotype, and markers of an inflammatory state, which are not recovered on return to a normal daily step count in healthy older adults. Ultimately, proactive interventions such as exercise or pharmaceuticals may be required to reverse decrements in metabolic health with transient periods of physical inactivity in this population.

Support or Funding Information

Supported by the Canadian Diabetes Association
Post‐ or Pre‐Injury Concentric Contractions Do Not Reduce Eccentric‐Contraction‐Induced Myofiber Damage in a Murine Model of Dysferlin‐linked Muscular Dystrophy
Authors: Begam Morium , Collier Alyssa F , Basel Chantel A , Blackmer Jacob M , Hass Joshua J , Konja Jasmine T , Samojedny Amber L , Roche Joseph A
Published: November 2023

Background

Concentric contractions have been shown to reduce delayed onset muscle soreness following eccentric contractions in healthy human subjects. Dysferlin‐deficient muscle undergoes severe delayed onset myofiber damage over several days after eccentric contractions. The goal of our study was to test the hypothesis that concentric contractions reduce eccentric‐contraction‐induced myofiber damage in dysferlin‐deficient muscle.

Methods

We studied the tibialis anterior (TA) muscle in 3–4 month old, male, dysferlin‐null BLAJ mice. We elicited TA muscle contractions by depolarizing the fibular nerve with an electrical stimulator; and used a custom‐built dynamometer to measure contractile torque and expose the TA muscle to eccentric contractions (one bout; 40 repetitions; 90–160 □ plantarflexion superimposed on maximal tetany of ankle dorsiflexors). We exposed one group of mice to a single bout of 20, maximal, unloaded concentric contractions soon after eccentric contractions (ConEx after EccEx; N = 4 mice); exposed a second group to 6 sessions of maximal, unloaded concentric contractions (40 repetitions/session) over 2 weeks (ConEx before EccEx; N = 4); and designated a third group as controls (Untreated; N =4). We studied contractile torque before, immediately after, and 3 days after eccentric contractions. We collected TA muscles 3 days after eccentric contractions to histologically assess myofiber damage through hematoxylin and eosin staining, and through immunoglobulin‐G (IgG) and desmin immunofluorescent labeling.

Results

Summarized in Table 1.

Conclusion

Neither post‐ nor pre‐injury concentric contractions reduce eccentric‐contraction‐induced myofiber damage in dysferlin‐deficient skeletal muscle.

Support or Funding Information

Funded by a Faculty Startup Package to JAR. Dysferlin‐null BLAJ mice were a kind gift from the Jain Foundation Inc.

Physiological and Histological Data

Outcome Measure
Untreated (Mean ± S.E.M.)
ConEx after EccEx (Mean ± S.E.M.)
ConEx before EccEx (Mean ± S.E.M.)
Statistically Significant Difference

Torque ‐ Immediately After Eccentric Contractions (% Pre)
52 ± 8
46 ± 1
49 ± 2
No

Torque ‐ 3 Days After Eccentric Contractions (% Pre)
37 ± 9
34 ± 1
38 ± 2
No

% Damaged Fibers ‐ 3 Days After Eccentric Contractions (H&E Staining)
42 ± 3
44 ± 2
41 ± 2
No

% IgG+ and Desmin‐Fibers ‐ 3 Days After Eccentric Contractions
40 ± 3
43 ± 3
38 ± 3
No
Chronic Heat Shock Stress Impairs Myoblast Proliferation and Differentiation
Authors: Bolus Daniel , Shanmugam Gobinath , Narasimhan Madhusudhanan , Soorapppan Rajasekaran Namakkal
Published: November 2023

Heat‐related illness and injury are becoming a growing safety concern for the agricultural, construction workers, miners, firefighters, manufacturing workers and other outdoor workforces who are exposed to heat stress (HS) in their routine lives. A primary response by a cell to an acute HS exposure is the induction of heat‐shock proteins (HSPs) that chaperones and facilitates cellular protein folding and remodeling processes.. Nonetheless, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast‐myotube model system of C2C12 cells have yet to be elucidated fully. In C2C12 myoblasts, we found that robust HS (43°C, dose/time) significantly decreased the proliferation by 25% on day 1 and day 2 of HS that was further decreased to 35% on day 3 of HS. This was accompanied by an accumulation of cells at S and G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. In addition, the repeated HS induced no apparent changes in apoptosis (markers) and cell death as determined by FACS analysis. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43°C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on day 2 and 3 maintained the elevated levels of HSPs than the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS compared to control cells. This was associated with significantly higher levels of HSPs and reduced expression of myogenin, Myh2, and MyoD, the genes involved in the differentiation process. Finally, the scratch assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated exposure of HS may perturb the active process of proliferation, motility and differentiation and affect the repair mechanisms in an in vitro murine myoblasts‐myotube model.

Support or Funding Information

These studies were supported by awards/research funds from the U.S. National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (R01HL118067), NIH National Institute on Aging (1R03AG042860‐01), the University of Utah, and the University of Alabama at Birmingham startup funds (to N.S.R.).
Morphological regeneration and functional recovery of neuromuscular junction after ischemic/reperfusion injury induced by tourniquet in mouse hind limb
Authors: Tu Huiyin , Zhang Dongze , Muelleman Robert L , Wadman Michael C , Li Yu‐Long
Published: November 2023

Background

Tourniquet‐induced ischemia and subsequent reperfusion cause serious acute ischemia‐reperfusion (IR) injury in the neuromuscular junction (NMJ) and skeletal muscle. These injuries are associated with permanent neurological deficiency, and long‐term skeletal muscle contractile dysfunction, even complete limb paralysis. Monitoring structural and functional repair of the NMJ and skeletal muscle after tourniquet‐induced IR is beneficial to exploring the mechanisms responsible for tourniquet‐induced IR injury and finding effective therapeutic interventions. Here, we observed long‐term morphological and functional alterations of the NMJ in tourniquet‐induced hindlimb IR.

Method

Unilateral hindlimb of C57/BL6 mice was subjected to 3 hours of ischemia by placing an orthodontic rubber band and followed different periods of reperfusion (1 day, 3 days, 1 weeks, 2 wekks, 4 weeks, and 6 weeks). At each time point of reperfusion, the gastrocnemius muscle was isolated for measurement of the NMJ morphology and function. Motor nerve terminals and nicotinic acetylcholine receptors (nAChRs) in the NMJ were measured by immunofluorescence staining. The end plate potential (EPP) was recorded by the intracellular recording technique.

Results

In the NMJ of sham mice, the structure of the nAChR clusters remained intact pretzel‐like shape and motor nerve terminals innervated the nAChR clusters to form synapses for signal transmission. The number of fragments in one nAChR cluster was 1.83 ± 0.07 and all nAChR clusters were innervated by motor nerve terminals (100%). The amplitude of EPPs was 29.84 ± 1.02 mV. At 1 day and 3 days of reperfusion, the motor nerve terminals disappeared, the AChR clusters kept normal shape, and no EPPs were recorded. From 1 week to 6 weeks of reperfusion, the motor nerve terminals gradually reinnervated (46.88%, 88.05%, 99.2%, and 100% for 1, 2, 4, and 6 weeks of reperfusion, respectively). However, the AChR clusters were gradually fragmented and the number of fragments in one nAChR cluster increased with longer period of reperfusion (2.92 ± 0.14, 5.50 ± 0.30, 8.94 ± 0.67, and 6.88 ± 0.50 for for 1, 2, 4, and 6 weeks of reperfusion, respectively; p<0.05 vs. sham). The amplitude of EPPs also increased gradually with longer period of reperfusion, but the amplitude of EPPs at 6 weeks of reperfusion didn't restore towards the level seen in sham mice (13.92 ± 1.11 mV, p<0.05 vs. sham).

Conclusion

Tourniquet‐induced ischemia and subsequent reperfusion impaired the NMJ. The morphological change of motor nerve terminals was faster than that of the nAChR clusters. The slow recovery of fragmented nAChR clusters could attenuate neuromuscular transmission.
Wnt Is Upregulated Following Skeletal Muscle Injury.
Authors: Keoonela Samantha , McCormick Skyler , Carpenter April
Published: November 2023

Wnt signaling is implicated in a variety of developmental and regeneration processes. In particular, canonical wnt signaling is upregulated following skeletal muscle injury in mice. There are currently 19 recognized wnt ligands with overlapping function. It is unknown which of the wnt ligands are expressed and the time course of their expression following injury and subsequent repair. To investigate the upregulation of wnt message following skeletal muscle injury, we injected the tibialis anterior of male mice with either saline control, or 1.2% barium chloride. Using qPCR we analyzed the message for wnts days 2,7 and 14 following muscle injury. mRNA for all 19 of the wnt proteins was absent in saline injected muscles at days 2, 7 and 14 days post‐injection. However, day 2 post‐BaCl2 injection, message for 10 of the 19 wnt ligands (Wnts 2, 2b, 3, 4, 5a, 6, 9, 10b, 11 and 16) was present. Day 7 post‐BaCl2 injection, upregulation of mRNA for 6 wnt ligands (Wnts 2, 2b, 4, 6, 11 and 16) was maintained. Message for wnt proteins was absent during the late phase of muscle regeneration (14 days post‐BaCl2 injection). These data suggest that wnt signaling during the early stages of inflammation following tissue injury may be important for the early phases of muscle repair. Studies are ongoing to determine the cellular source of the increased wnt expression and causative studies of mutant mouse models will determine the impact of wnt signaling in muscle repair.
The Dunkin‐Hartley Guinea Pig as a Novel Model for Sarcopenia
Authors: Musci Robert V , Wolff Christopher A , Reid Justin R , Laurin Jaime L , Peelor Frederick F , Konopka Adam R , Santangelo Kelly S , Miller Benjamin F , Hamilton Karyn L
Published: November 2023

Increasing age is a primary, non‐modifiable risk factor for many chronic diseases, including sarcopenia. Sarcopenia, or age‐related muscle loss, requires an imbalance of protein turnover where protein breakdown exceeds protein synthesis. The purpose of this investigation was to characterize age‐related changes in skeletal muscle in the Dunkin‐Hartley (DH) guinea pig to evaluate its potential as a model of accelerated skeletal muscle aging. DH and control Strain 13 (S13) guinea pigs at 5, 9, and 15 months (mo) of age were injected with an intraperitoneal bolus of the stable isotope deuterium oxide (D2O) and provided with 8% D2O‐enriched drinking water for 2 weeks to evaluate gastrocnemius protein synthesis rates in subcellular fractions enriched with mitochondrial, cytosolic, myofibrillar, or collagen proteins. In muscle from 5 and 15 mo guinea pigs we characterized myofiber size distribution in slow twitch (ST) and non‐slow twitch (FT) fibers using immunofluorescent staining with laminin and ST myosin heavy chain‐1. Sirius Red staining was used to assess collagen composition of total protein content in muscle tissue. 15mo DH had lower protein synthesis rates (%/day) compared to 5mo DH in mitochondrial (5mo 2.254±0.241, 15mo 1.606±0.280 P<0.05), cytosolic (5mo 2.548±0.187, 15mo 1.853±0.313 P<0.05), and myofibrillar (5mo 2.455±0.320, 15mo 1.797±0.315 P<0.05) enriched fractions of the gastrocnemius. There were no changes in protein synthesis rates observed between 5mo and 15mo S13. By 9mo, collagen synthesis was decreased compared to 5 month in both DH (5mo 0.592±0.152, 9mo 0.301±0.062 P<0.05) and S13 (5mo 0.622±0.126, 9mo 0.323±0.090 P<0.05). Gastrocnemius collagen content (%) was unchanged with age in both strains (DH: 5mo 3.99±0.16, 15mo 3.42±0.21 P>0.05 S13: 5mo 3.92±0.33, 15mo 3.95±0.06 P>0.05). Data (n=15) indicate an age‐related increase in gastrocnemius myofiber ST percentage in the DH strain (5mo 14.77±2.98%, 15mo 27.62±8.07%) as well as a shift in myofiber size distribution to smaller ST and FT fibers. To summarize, we observed decreased skeletal muscle protein synthesis at 15mo compared to 5mo in DH but not S13 guinea pigs. We also observed a shift to smaller myofiber size in DH. Altogether, DH guinea pigs demonstrated characteristics of age‐related sarcopenia while S13 did not by 15mo. Our results suggest that the DH strain might be a non‐transgenic model of accelerated aging that could be used to measure sarcopenia in vivo.
A single bout of heat stress treatment increases Nrf2 and its target genes in mouse skeletal muscle
Authors: Tamura Yuki , Kitaoka Yu , Nakazato Koichi , Hatta Hideo
Published: November 2023

In recent years, we have reported that heat stress induces various skeletal muscle adaptations (e.g. mitochondria, autophagy, unfolded protein response). In this study, we explored further possibilities of muscle adaptations by heat stress treatment using transcriptome approach and subsequent bioinformatics analysis. Immediately after treatment and three hours after a single bout of heat stress treatment (exposing mouse into a hot environment chamber; 40°C, 30 min), gastrocnemius muscles were collected and then examined changes in over 39,000 genes expression using Affimetrix microarray GeneChip. Consequently, we analyzed with bioinformatics algorithms TFactS and BioCarta to predict activated transcriptional factors and activated pathways, respectively. These computational analyses based on transcriptome data indicated that heat stress activates Nrf2 (Nfe2l2), a master transcriptional factor of antioxidant response, in skeletal muscle (TFactS: P<0.05; BioCarta: Fold Enrichment=7.2, P<0.05). We further confirmed by qPCR that heat stress increased Nrf2 (+39.4%, P<0.05) and its target genes such as Cat (+30.9%, P<0.05), Hmox1 (+182.6%, P<0.05), Gclc (+53.8%, P<0.05), Gclm (+32.1%, P<0.05), Gpx1 (+23.7%, P=0.07), Mt1a (+251.6%, P<0.05) and Sod1 (+27.8%, P<0.05) at 3h after treatment. Notably, there were non‐detectable changes in oxidative stress marker (4HNE‐conjugated protein) and gene expression of Nrf2‐independently inducible antioxidant enzyme (Sod2) at both immediately post‐treatment and 3h after treatment. Our observations suggest that the physiological significance of Nrf2 activation by heat stress may be other than defending oxidative stress.

Support or Funding Information

This study was supported by Grant‐in‐Aid for JSPS Research Fellow.
Hyperthermia and extracellular hyperosmolality affect sarcolemmal damage and protein oxidation in mammalian skeletal muscle in vitro
Authors: Laitano Orlando , Mattingly Alex J. , Robinson Gerard P. , Garcia Christian K. , Clanton Thomas L.
Published: November 2023

In the US Armed Services, the incidence of rhabdomyolysis is 5–7 fold higher in peak summer months, suggesting that muscle membrane injury could be amplified by heat or to secondary dehydration. In this study we tested the extent each of these variables influences damage by evaluating sarcolemmal integrity and ROS‐induced protein oxidation. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected from 24 adult male C57BL6J mice and placed at optimal length in tissue baths containing 4 ml of Krebs (95%O2/5%CO2). Muscles were intermittently stimulated to perform non‐fatiguing twitches for 30 min. Buffer temperature and osmolality were manipulated to provide four experimental conditions: normothermia/isotonic (35°C and 285 mOsm/kg), normothermia/hypertonic (35°C and 300 mOsm/kg), hyperthermia/isotonic (41°C and 285 mOsm/kg), and hyperthermia/hypertonic (41°C and 300 mOsm/kg). We quantified the integrity of the sarcolemma using both procion orange leakage into the fibers and a fluorometric assay to detect leakage out of double‐stranded cell‐free DNA (cfDNA). To assess protein oxidation, we performed immunoblot assays for protein carbonyl derivatives. For SOL we did not observe a significant effect of hyperthermia and/or hyperosmolality on procion orange leakage, but we observed an increase in cfDNA (p = 0.03) and protein carbonyls with hyperthermia (p = 0.02). Interestingly, these effects were absent when hyperosmolality was superimposed on hyperthermia. For EDL, an increase in procion orange leakage was observed with hyperthermia alone (p = 0.04) and when hyperthermia and hyperosmolality were combined (p = 0.03), but the combination did not elicit changes in cfDNA in the buffer. EDL protein carbonyls were independently augmented with hyperthermia (p = 0.01) and hyperosmolality (p = 0.02), but also did not increase when hyperthermia was combined with hyperosmolality. In conclusion, hyperthermia caused sarcolemma damage in EDL muscle, both alone and when combined with hyperosmolality. SOL was more resistant to hyperthermia‐induced procion orange leakage, despite an increase release of extracellular cfDNA. Furthermore, physiological increases in extracellular osmolality prevented protein oxidation in hyperthermia in both muscle types. These observations suggest that different muscle types may respond differently to hyperthermic and osmotic challenges. The compensatory effect of hyperosmolality on hyperthermia‐induced oxidative stress is consistent with a role for ROS production in compensatory responses to cell swelling in the heat.

Support or Funding Information

Supported by the BK and Betty Stevens Endowment and the Brazilian Council for the Development of Science and Technology (CNPq no. 249094/2013‐4).
H2S MEDIATES SENSORY LONG‐TERM FACILITATION OF THE CAROTID BODY
Authors: Peng Ying‐Jie , Yuan Guoxiang , Khan Shakil , Nanduri Jayasri , Kumar Ganesh , Snyder Solomon , Prabhakar Nanduri
Published: November 2023

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. Rodents treated with CIH exhibit hypertension and increased sympathetic nerve activity, and these effects were attributed to carotid body (CB) chemoreflex. CIH leads to CB activation manifested by augmented sensory response to hypoxia, and sensory long‐term facilitation (LTF). The purpose of the present study is to determine the mechanisms underlying CB activation by CIH. Recent studies suggest that the gasotransmitter H2S mediates CB activation during acute hypoxia. Since CIH increases H2S production from the cystathionine‐ϒ‐lyase (CSE) in the CB through ROS signaling, we hypothesized that H2S mediates the CB activation by CIH. Experiments were performed on adult rats, CSE‐null and wild‐type mice treated with 10 days of CIH. CIH treated CBs displayed augmented sensory response to hypoxia, and sensory LTF in rats and wild‐type mice. CB activation by CIH was completely absent in rats treated with L‐Propargylglycine (L‐PAG) an inhibitor of CSE and in mice deficient in CSE. CIH treated rats and wild‐type mice showed hypertension, increased sympathetic nerve activity, and elevated plasma catecholamine levels ‐ all these effects were remarkably absent in L‐PAG treated rats and CSE null mice. These findings suggest that ROS‐dependent activation of CSE‐derived H2S synthesis mediates CB response to CIH and the ensuing hyperactive CB chemoreflex contributes to CIH‐induced sympathetic activation and hypertension.

Support or Funding Information

Supported by NIH‐PO1‐HL90554
BLOCKADE OF NMDA RECEPTORS IN THE INSULAR CORTEX OF SPONTANEOUSLY HYPERTENSIVE RATS RESULTS IN LARGE FALL IN ARTERIAL PRESSURE
Authors: Marins Fernanda Ribeiro , Iddings Jennifer A , Filosa Jessica A , Fontes Marco Antônio Peliky
Published: November 2023

We have recently shown that the intermediate region of the posterior insular cortex (intermediate IC) of spontaneously hypertensive rats (SHR) presents marked alterations in the neurovascular unit. Immunofluorescence data also shows increased NMDA receptor (NMDAR) density within the intermediate IC of SHR. In addition, stimulation of NMDAR in the intermediate IC of SHR results in higher amplitude responses of renal sympathetic activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) when compared to normotensive controls (WKY). The aim of this study was to determine the sympathetic and cardiovascular changes evoked by blockade of endogenous NMDA in the intermediate IC of SHR. Under urethane anesthesia (1.4 g/Kg, i.p.), bilateral microinjection of the NMDA receptor antagonist, AP‐5 (5mM/100nL), was performed in the intermediate IC of SHR (n=4) or WKY (n=6) with simultaneous recording of RSNA, HR and MAP. When compared to WKY, SHR presented higher baseline levels (measured before AP‐5 injection) of sympathetic nerve activity discharge (SHR 182±6 vs. WKY 122±4 discharges/second, P<0.0001) and MAP (SHR 144±6 vs. WKY 91±3 mmHg, P<0.0001). No differences were observed in baseline HR (SHR 368±12 vs. WKY 383±5 bpm, P= 0.3761). Bilateral injection of AP‐5 into the intermediate IC of SHR evoked a marked decrease in MAP (Δ= −31.7±4.8 mmHg, P=0.0004) accompanied by substantial decreases in RSNA (Δ= −16 ± 3.6 %, P=0.0114) and HR (Δ= −18.1±9.5 bpm, P =0.0381). No significant changes were observed after AP‐5 injection into the intermediate IC of WKY (Δ RSNA= 1±4.1 %, P=0.3866, ΔMAP= −1.3±1.4 mmHg, P=0.5010 and ΔHR= 0.6±0.8 bpm, P =0.4411). Taken together with our previous findings, the results suggest that activation of NMDA receptors in the intermediate insular cortex contribute to an enhanced sympathetic vasomotor tone in spontaneously hypertensive rats. CNPq, FAPEMIG, CAPES, NHLBI.

Support or Funding Information

Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brazil (PQ CNPQ 306000/2013‐0), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), INCT Nanobiofar, and CAPES (99999.003909/2015‐08 Programa PDSE), National Heart, Lung, and Blood Institute (NHLBI) of the NIH (R01 HL089067‐02 to JAF).
Opposite Arterial Pressure Responses to Renal Afferent Nerve Stimulation in Unanesthetized Versus Anesthetized Rats: Implications for the Neural‐Renal Axis and Hypertension
Authors: Banek Christopher T , Osborn John W
Published: November 2023

Neural control of the kidney remains a primary focus of treatment of cardiovascular and renal disease, including hypertension (HTN). Moreover, recent studies support a role of renal afferent nerves in the modulation of sympathetic nerve activity (SNA), and in turn, arterial pressure (AP). We recently reported resting afferent nerve activity (ARNA) is elevated in the DOCA‐salt hypertensive rat, and that targeted afferent renal nerve ablation attenuates this model of hypertension (Banek et al. Hypertension 2016). These findings suggest that increased ARNA contributes to the pathogenesis of HTN. Previous investigations of the responses of AP and SNA to renal afferent nerve stimulation have been conducted in anesthetized animals have reported a sympatho‐inhibitory response. The present study was designed to measure the acute AP response to increased ARNA in anesthetized and unanesthetized preparations. Male Sprague Dawley rats (300–400g) underwent acute renal afferent stimulation while under 2% isoflurane anesthesia (ISO; n=12) or in the absence of anesthesia following a cortical decerebration (DEC; n=12). Animals were instrumented to measure AP, heart rate (HR), and ARNA. To activate renal afferent nerves, a ureteral catheterization was performed to perfuse the renal pelvis with vehicle 0.9% saline [VEH] or with the TRPV1‐agonist 50μM capsaicin (CAP). Following instrumentation and a 30‐minute stabilization period, ARNA and cardiovascular responses to VEH or CAP stimulation were recorded. Peak responses in ARNA, AP, and HR were quantified and compared between ISO vs. DEC preparations by a Student's t‐test (α=.05). ARNA responses were calculated as percent change from baseline (%ARNA). Data presented as mean±SEM. There was no difference in resting ∫ARNA, AP, or HR prior to afferent stimulation between ISO and DEC groups. VEH perfusion of the renal pelvis elicited no response in both ISO and DEC groups. In contrast, CAP administration resulted in marked increase in %ARNA that was similar in magnitude in both groups (ISO 214±31% vs. DEC 239±40%). More importantly, whereas the CAP‐induced increase in ARNA elicited a depressor response in ISO rats (−17±6mmHg), CAP evoked an opposing pressor response in DEC rats (+54±9mmHg). These findings suggest the existence of a renal afferent nerve‐dependent sympatho‐excitatory reflex in the rat, and anesthesia may directly interfere in this signaling pathway. It is important to note it is unknown whether this acute reflex translates to long‐term regulation of SNA and AP. While the anatomical and biological mechanisms of this reflex remain rudimentary, further studies are underway to chronically monitor afferent nerve activity and blood pressure in conscious freely moving rats to elucidate this important neural‐renal axis.

Support or Funding Information

NIH 5R01HL116476; NIH 2T32HL7741
High fat diet increases salt sensitivity and promotes metabolic disorder‐independent hypertension in Dahl salt sensitive rats
Authors: Fernandes Roxanne , Bonilla Patricia A Perez , Garver Hannah , Galligan James J , Fink Gregory D , Xu Hui
Published: November 2023

Salt sensitivity of blood pressure is common in obesity associated hypertension and may have a neurogenic basis. We and others have previously reported that high fat (HF) diet promotes hypertension in Dahl salt sensitive (SS) male rats ingesting a “normal” salt diet (0.33% NaCl). It is unclear if a low salt intake would reduce blood pressure in this model of obesity‐associated hypertension. Therefore in the current study Dahl SS rats were fed a normal fat (NF, 10% kcal from fat) and HF (60% kcal from fat) diet with different salt contents for 24–26 weeks after weaning (3 weeks of age). In rats fed a diet containing 0.24–0.33% NaCl (the normal salt content (NS) in rodent diets), HFNS (n=9) rats had a much higher MAP than NFNS rats (n=10) at the end of the study (158±1 mmHg versus 130±1 mmHg, P<0.05), confirming our previous findings. However, in low salt fed rats (LS, 0.1% NaCl), MAP at the end of the study was only slightly higher in HFLS (n=4) than NFLS (n=5) rats (131±1 mmHg vs 121±3 mmHg, P<0.05). Body weight was not significantly greater, but visceral adipose accumulation was higher, in HFNS and HFLS rats compared to NFNS and NFLS fed rats, respectively. HF fed rats did not show significant alterations in plasma levels of fasting glucose, insulin, leptin, aldosterone and serum creatinine compared to NF rats. Only HFNS rats showed hyperlipidemia and only NFLS rats showed lower plasma urea BUN levels than the other groups of rats. HFNS rats had a larger depressor response to ganglion blockade than did NFNS rats, but this was not observed in HFLS rats compared to NFLS animals. HF diet did not affect the mesenteric arterial (MA) contractile responses to neurotransmitters (norepinephrine and ATP), but MA from HFNS rats exhibited significantly increased wall‐thickness (and cardiac hypertrophy) compared to NFNS rats. Morphological studies did not reveal greater fibrosis in adventitia of mesenteric, intrarenal or coronary arteries in HF rats, suggesting arterial hyperplasia accounted for arterial remodeling in HFNS rats. Our studies suggested that obesity‐associated hypertension in Dahl SS rats is salt‐sensitive and neurogenic, but not associated with obvious metabolic disorders.

Support or Funding Information

Supported by NIH2P01HL070687‐11A1
Chronic Renal Medullary Infusion of Interleukin‐1 Receptor Antagonist Delays the Onset of Salt Sensitive Hypertension in Uninephrectomized Dahl SS Rats
Authors: Dasinger John Henry , Abais‐Battad Justine M , Lund Hayley , Fehrenbach Daniel J , Mattson David L
Published: November 2023

The Dahl SS rat is a well‐established model of salt‐sensitive hypertension and renal disease with the immune system contributing in the pathology of the disease. Recent work from our laboratory demonstrated that in response to a high salt challenge, in addition to increases in blood pressure and urinary albumin excretion, there is an upregulation of the Nod‐like receptor protein‐3 (NLRP3) inflammasome in the renal medulla. This upregulation of the NLRP3 inflammasome leads to a subsequent increase in interleukin 1 beta (IL‐1β) production. This proinflammatory cytokine has been shown to be elevated in hypertension in humans making it a potential therapeutic target for salt sensitive hypertension. Therefore, we tested the hypothesis that chronic renal medullary infusion of IL‐1 receptor antagonist (IL‐1RA) would attenuate the development of salt sensitive hypertension in the Dahl SS rat. Animals underwent a right uninephrectomy at 7 weeks of age to avoid compensatory changes in blood pressure from the contralateral kidney. After a week of recovery, the left femoral artery was cannulated to measure mean arterial pressure (MAP) while a chronically implanted medullary interstitial catheter was placed in the left kidney for continuous infusion IL‐1RA (600μg/day, n=5) or vehicle (0.9% NaCl saline, n=6). MAP was measured during a 4‐day period while animals were maintained on 0.4% NaCl chow then switched to a high salt (4.0% NaCl) challenge for a 10‐day. MAP did not differ between saline and IL‐1RA treated animals during the 0.4% diet period (127±3 mmHg vs 121±4 mmHg, P>0.05). However, following a 10‐day challenge of 4.0% NaCl diet, MAP was significantly reduced in the IL‐1RA treated rats compared to the saline‐treated rats (157±9 mmHg vs 176±5 mmHg, P<0.05). Despite seeing a reduction in MAP, urinary albumin (118±18 vs 126±10 mg/day) and protein (284±28 vs 277±19 mg/day) excretion in IL‐1RA treated did not differ from saline‐treated rats. Furthermore, when medullary IL‐1β levels were measured by ELISA, there was no difference between the IL‐1RA treated rats compared to the saline‐treated rats (76±25 pg/mL vs. 83±11 pg/mL). In contrast to the effects of intarenal infusion, systemic administration of the same dose of IL‐1RA did not alter salt‐sensitive hypertension compared to vehicle‐treated control SS (132±8mmHg vs 141±mmHg, n=5–6/group), demonstrating that the effects observed with medullary interstitial infusion are due to intrarenal effects. These data indicate that blockade of the IL‐1 receptor in the renal medulla attenuates salt‐sensitive hypertension in the Dahl SS rat. Further studies are required to understand the mechanism by which renal medullary inhibition of IL‐1β results in reduction in blood pressure in response to a high salt diet.

Support or Funding Information

Supported by DK96859, HL116264, and 15SFRN2391002.
Sex‐Dependent Regulation of Blood Pressure by the Circadian Clock Protein Per1
Authors: Solocinski Kristen , Cheng Kit‐Yan , Lynch I. Jeanette , Cain Brian D. , Wingo Charles S. , Gumz Michelle L.
Published: November 2023

The circadian clock is integral to the maintenance of daily rhythms of many physiological outputs including blood pressure. Our lab has demonstrated the importance of the clock protein Per1 to blood pressure regulation. Previously, we determined the effects of a high salt diet plus mineralocorticoid on Per1‐mediated blood pressure regulation in male C57BL/6 mice (Solocinski et al. Acta Physiol 2016). As expected, mean arterial pressure (MAP) in wild type mice was not affected by a high salt diet (4%)(HS) plus injection with the long‐acting mineralocorticoid deoxycorticosterone pivalate (DOCP). However, Per1 knockout mice exhibited non‐dipping hypertension in response to this treatment. The goal of the present study was to determine the role of Per1 in the regulation of MAP in female mice. For the first time, we demonstrate that loss of a circadian clock protein in female mice results in increased MAP relative to littermate wild type control mice on a C57BL/6 background. These mice were subjected to HS/DOCP treatment as well. In contrast to what we previously observed in male wild type mice, female wild type mice did experience a significant increase in MAP in response to HS/DOCP (7.35 ± 1.00 mmHg p < 0.0001). The Per1 knockout female mice also experienced an increase (8.29 ± 1.17 mmHg, p < 0.0001). Neither genotype experienced changes in the day/night MAP ratio in response to HS/DOCP. Together these data demonstrate that Per1 is important for MAP regulation in female animals and that this regulation appears to be salt‐ and mineralocorticoid‐independent. This effect is distinct from the non‐dipping hypertension seen in male Per1 knockout mice, demonstrating that female sex appears to be protective against Per1‐ mediated non‐dipping hypertension in response to HS/DOCP. Together these data support an important role for the circadian clock protein Per1 in the modulation of blood pressure and suggest that Per1 acts in a sex‐dependent manner in the regulation of cardiovascular rhythms.

Support or Funding Information

NIH NIDDK

UF Department of Medicine, Division of Nephrology

American Society of Nephrology

UF Hypertension CenterT32 Grant # 2T32HL083810

American Heart Association
PVN inflammation ‐ the driver of sympathetic outflow and sodium retention in salt sensitive hypertension?
Authors: Wainford Richard
Published: November 2023

Background

We have reported central Gαi2 protein down regulation evokes sympathetically mediated salt sensitive hypertension in multiple salt‐resistant rat phenotypes. Interestingly, several laboratories report that PVN inflammation is a mechanism that contributes to increased sympathetic nervous system (SNS) activity and HTN.

Aim

We hypothesize PVN inflammation contributes to increased SNS activity and salt sensitive HTN following central Gαi2 protein down regulation in the Sprague‐Dawley rat.

Methods

Male Sprague‐Dawley (SD) rats receiving a continuous i.c.v. control scrambled (SCR) or targeted Gαi2 oligodeoxynucleotide (ODN) infusion (25μg/5μl/day) were fed a 0.6% (NS) or 4% NaCl (HS) diet for 7 days (N=4/group). On day 7 24h sodium balance, basal MAP, plasma NE content (via ELISA) and PVN specific mRNA levels of IL1β, IL6, IL10 and TNFα (via RT‐PCR) were assessed. Additional groups of rats received a SCR or Gαi2 ODN and minocycline (120 μg/day) co‐infusion and a 7‐day HS diet.

Results

In Male SD rats CNS Gαi2 down regulation, but not control SCR ODN treatment, results in HS evoked HTN, sympathoexcitation, sodium retention, increased PVN mRNA levels of the pro‐inflammatory cytokines (PIC) IL1β, IL6 and TNFα and a decrease in PVN anti‐inflammatory 1L10 mRNA expression. ICV minocycline (anti‐inflammatory antibiotic) co‐infusion did not alter the physiological responses to HS intake in SCR ODN infused rats but abolished the increase in HS evoked PVN PIC mRNA levels and attenuated HTN, sympathoexcitation and sodium retention following CNS Gαi2 down regulation.

Conclusion

Our data suggests that PVN inflammatory mechanisms contribute to the pathophysiology of CNS Gαi2 dependent salt sensitive HTN through influences on central sympathetic outflow and sodium homeostasis. These data broaden the settings in which PVN inflammation influences blood pressure to include salt sensitive HTN.

Support or Funding Information

This work was supported by NIH grants R01HL107330 and K02HL112718 and AHA 16MM32090001 to RDW.

ICV SCR ODN INFUSION (25μg/5μl/day)
ICV Gai2 ODN INFUSION (25μg/5μl/day)

Normal Salt (0.6% NaCl)
High Salt [HS] 4% NaCl
HS + ICV MINO (120μg/5μl/day)
Normal Salt (0.6% NaCl)
High Salt [HS] 4% NaCl
HS + ICV MINO (120μg/5μl/day)

MAP (mmHg)
123±4
121±3
119±3
118±4
144±3*
130±4*#

Plasma NE (nmol/l)
62±7
38±4*
42±5*
59±5
92±7*
62±6#

24h Na+ balance (mEq)
0.3±0.1
0.4±0.1
0.3±0.1
0.3±0.1
1.8±0.3*
0.9±0.2*#

IL‐1β (fold increase)
0.9±0.2
0.8±0.1
1.1±0.2
1.1±0.2
2.4±0.4*
1.3±0.3#

IL‐6 (fold increase)
1.0±.2
0.9±0.1
1.1±0.1
1.0±0.1
2.2±0.3*
1.2±0.2#

TNFα (fold increase)
1.1±0.1
1.0±0.2
1.0±0.1
1.3±0.2
2.3±0.5*
1.1±0.2#

IL‐10 (fold increase)
1.0±0.1
1.3±0.1
1.3±0.2
1.1±0.2
0.4±.0.1*
1.1±0.2#

p<0.05 vs. respective normal salt (0.6% NaCl) group;

p<0.05 vs. respective high salt (4% NaCl) group (N=4/group ± SEM).

MINO = minocycline.
Maternal Hyperandrogenemia Causes Increased MAP and Proteinuria in Male Rat Offspring
Authors: Dalmasso Carolina , Maranon Rodrigo O. , Patil Chetan N. , Reckelhoff Jane F.
Published: November 2023

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting 6–10 % of women, and is associated with hyperandrogenemia, oligo/anovulation and polycystic ovaries. Many women with PCOS have difficulty getting pregnant, and thus this population often opts for in vitro fertilization. However, the offspring of PCOS women are predisposed to lower birth weight, metabolic disorders during childhood, and an increased risk for the daughters to develop PCOS. However, the mechanisms by which the offspring of women with PCOS develop negative health outcomes are unknown. Therefore, these studies tested the hypotheses that females of PCOS model in rats would develop symptoms of PCOS and the males would develop other cardiovascular symptoms such as elevated blood pressure. Female (F) and male (M) offspring of control (C) and PCOS (P) dams (SD; DHT 7.5mg/90d) were divided into groups: F‐C, F‐P, M‐C and M‐P. At postnatal day 2, 7, 15 and 21 there was a significant increase (p<0.05) in body weight in both F‐P and M‐P, compared to F‐C and M‐C, respectively. This trend reversed by week 17 with F‐P and M‐P have lower body weights (p<0.05) compared with F‐C and M‐C, respectively. Proteinuria, measured at week 24, was similar in F‐P vs. F‐C (p=NS), but was significantly higher in M‐P than M‐C (18.8±4.1 vs. 48.5±14.1 mg/d, p<0.05). Baseline mean arterial pressure (MAP, telemetry) was tended to be higher in F‐P vs. F‐C (F‐C= 109±5 mmHg vs. F‐P= 118±3 mmHg; p=NS). However, in males, MAP was significantly higher in M‐P than M‐C (M‐C= 113±2 mmHg vs. M‐P= 121±3 mmHg; p<0.05). To test whether exposure to hyperandrogenemia in utero would cause an exaggerated response to Angiotensin II (Ang II), male and female offspring were treated with enalapril (7d; drinking water; 250 mg/l) for 5 days, and then Ang II (150 mg/kg/d, sc minipump, 7 days). MAP decreased with enalapril similarly in F‐C and F‐P, and in M‐C and M‐P, and were similar among the groups (F‐C: −18÷4; F‐P: −22±5; M‐C: −16±1; M‐P: −18±1 mm Hg; p=NS for all groups), and maintained the difference in MAP in females, but not males. ANG II infusion increased MAP to similar levels in males and females: F‐C: 162±9; F‐P: 160±7 mm Hg (p=NS); M‐C: 162±6; M‐P: 171±1 mm Hg (p=NS). Thus male offspring of PCOS dams have an increase in MAP and more proteinuria, but similar responses to Ang II compared to controls. Female offspring do not develop a PCOS phenotype. Future studies will be necessary to determine exact mechanisms responsible for increase in blood pressure and renal injury.

Support or Funding Information

Supported by NIH‐R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM).