bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying Molecular Mechanisms and Biomarkers for Arterial Stiffness in Vitamin D Deficiency, Obese, and Diabetic Patients
Authors: Elmoselhi Adel , Seif Allah Mohamed , Bouzid Amal , Ibrahim Zeinab , Bajbouj khuloud , Abou Assaleh Rebal , Venkatachalam Thenmozhi , Siddiqui Ruqaiyyah , Khan Naveed , Hamoudi Rifat
Published: May 2023

Background and Aims: Patients who suffer from vitamin D3 deficiency, obesity, and diabetes mellitus (DM) are at higher risk of cardiovascular diseases (CVDs). Arterial stiffness is also shown to be increased and linked with those patients. In this study, we intended to identify circulating MicroRNAs (miRNAs) biomarkers and explore molecular mechanisms of arterial stiffness in those patients as early predictors of CVDs. Methods and Results: Twenty-three adult Emiratis patients were recruited and divided in the following groups: A control healthy group with vitamin D ≥ 20ng, and BMI ≤ 30 (C1 group =11 individuals); a second group with vitamin D deficiency (Vit D level ≤ 20 ng) and obesity (BMI ≥ 30) (A1 group = 9 patients); and a third group with vitamin D deficiency, obesity, plus Diabetes Mellitus (DM) (A2 group = 3 patients). Pulse wave velocity (PWV) was measured and the whole transcriptome analysis with qPCR validation for miRNA in plasma samples were tested. PWV relative to age was significantly higher in A1 and A2 groups 19.4 ± 4.7 m/s and 18.3 ± 1.3 m/s respectively, compared to the control group 14.7 ± 2.1 m/s (p < 0.05). Whole RNA-Sequencing revealed miR-182-5p; miR-199a-5p; miR-193a-5p; and miR-155-5p were differentially over-expressed (logFC > 1.5) in A1 and A2 groups. Additionally, the expression of the following oxidative stress and inflammatory biomarkers were significantly (p <0.05) higher in the A1 group vs C1 group: HIF-1alpha, NOX-I, NOX-II, cytokine IL-1b, IL-6, IL-8, IL-10, and VEGF. Conclusion: We identify 4 miRNAs for arterial stiffness in patients with vitamin D deficiency, obesity and DM. Furthermore, several oxidative stress and inflammatory genes and biomarkers seem to underlie the culprits of arterial stiffness in vitamin D deficiency and obese patients. Those biomarkers could serve as diagnostic and therapeutic targets for the early prevention of CVDs.

Sheikh Hamdan Bin Rashid Al Maktoum Award for Medical Sciences, Ref: MRG/38, and Targeted Research Grant from University of Sharjah, Office of Research and Graduate Studies.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Effect of doxycycline in autonomic modulation of the heart, assessed by heart rate variability, in rats with myocardial infarction
Authors: Durand Marina , Sanches Elen , Costa Lucas , Silva Carlos , Buzinari Tereza , Salgado Helio , Fazan Jr Rubens
Published: May 2023

Treatment with doxycycline (DOXI) reduces ventricular remodeling and decreases the susceptibility of arrhythmias after myocardial infarction (MI), probably due to its inhibitory effect on metalloproteinases, inflammatory mediators, and oxidative stress. We evaluated the effect of DOXI on heart rate variability (HRV) in rats with MI. Infarcted rats (coronary ligation) or control animals (sham-operated), were treated with DOXI (30 mg/kg/day) or vehicle for 4 weeks (N=7-10/group). Rats were implanted with subcutaneous electrodes and, on the following day, had ECG recorded for 30 min. Series of RR intervals were generated to calculate HRV indices. DOXI-treated control rats had lower heart rate (HR) than vehicle-treated counterparts (327±7 vs. 352±7 bpm). Infarcted rats, treated or not, had similar heart rate (344±7 and 350±6 bpm). The standard deviation of RR intervals was similar among groups, but the differences between successive RR (RMSSD) were smaller in DOXI-treated sham-operated rats (4.0±0.5 vs. 6.3±0.9 ms). No differences were observed in RMSSD in rats with MI. Spectral analysis showed that the LF/HF ratio was lower in DOXI-treated control rats (0.32±0.05 vs. 0.62 ± 0.07). There was no difference in the LF/HF ratio in infarcted rats treated with DOXI or vehicle. The symbolic analysis, as described by Porta et al., showed a lower occurrence of 0V patterns (19±4 vs. 33±3%) and a higher 2UV (38±5 vs. 22±2%) in sham-operated rats that received DOXI. In rats with MI, DOXI symbolic indices were found to be similar. Our findings strongly suggest that 4-weeks of treatment with DOXI lead to a change in sympathovagal balance to the parasympathetic side. Curiously this effect was seen only in sham-operated but not in infarcted rats.

Funding Sources: Fundação de Amparo à Pesquisa do Estados de São Paulo - FAPESP, 2020/06043-7. Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq, Process 306994/2021-6.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Efferent network of Neuromedin B neurons in the retrotrapezoid nucleus in mice
Authors: Abbott Stephen , Berry Faye , Stornetta Daniel , Bayliss Douglas , Souza George
Published: May 2023

In the parafacial region of the medulla, the peptide Neuromedin B (NMB) is expressed in retrotrapezoid nucleus (RTN) neurons that are proposed to be central respiratory chemoreceptors. The efferent network of RTN Nmb neurons has been established in rats, but has not been examined in mice in detail. To map the downstream connections of RTN Nmb neurons we performed genetically-targeted tracing using novel Nmb-Cre knock-in mice. Adult Nmb-Cre mice were injected in the RTN unilaterally with a Cre-dependent AAV producing expression of red fluorescent protein (AAV-CreON-mScarlet). Expression of Nmb was identified by fluorescent in situ hybridization (FISH). Injections of AAV-CreON-mScarlet in the RTN (N=3) transduced 58 ± 11 % of Nmb-expressing neurons with a selectivity for Nmb neurons of 98 ± 1 %. Intensely labelled axons and terminals were observed in regions overlapping with the medullary and pontine respiratory network. We observe an intra-RTN network of fibers with close appositions between RTN Nmb neurons, as well as moderate projections to areas dorso-medial to the facial nucleus and medially adjacent to the locus coeruleus. There was a modest contralateral projection to the medullary respiratory network, but none to the pontine respiratory network. No labelled fibers were found rostral of the parabrachial nucleus. Anterograde tracing of RTN Nmb neurons was complemented using retrograde transport of Cre-recombinase (AAVrg-Cre) in R26-loxSTOPlox-L10-GFP mice (eGFP-reporter) mice combined with labelling of transcript for Nmb by FISH. Small volumes (3-6 nL) of AAVrg-Cre were unilaterally injected in the Pre-Bötzinger complex (PreBöt), lateral parabrachial nucleus (LPB), caudal ventral respiratory group (cVRG), and the nucleus of the solitary tract (NTS). We determined the proportion of total RTN Nmb neurons that were retro-labelled from each target (PreBöt- 75 ± 3%, n=3; LPB- 65 ± 3%, n=4; cVRG- 54 ± 6%, n=3; NTS- 38 ± 3%, n=3). This result suggests that RTN neurons have a high degree of collateralization within the medullary and pontine respiratory network. Injections of AAVrg-Cre in the PreBöt labelled 16 ± 11% of RTN Nmb neurons contralateral to the injection site. Collectively, these neuroanatomical observations show that the efferent network of RTN Nmb neurons broadly innervate and selectively the central respiratory network in alignment with prior descriptions in rat. Furthermore, we provide evidence for novel connectivity between RTN Nmb neurons. This anatomy supports the idea that RTN neurons generate a coordinated widespread excitation of the respiratory rhythm and pattern generating network in order to promote alveolar ventilation.

Funding from NIH-HLBI #HL148004

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The effect of chronic unpredictable stress on renal mitochondrial function in female mice
Authors: Frambes Noelle , Crockett Alexia , Stegmann Kyle , Churillo Amelia , Hollis Fiona , Spinale Francis , Wood Susan , Webb R , Ryan Michael
Published: May 2023

Background: Chronic stress can induce high blood pressure and lead to decreased renal function by mechanisms that remain unclear. The exposure of rodents to chronic unpredictable stress (CUS) is a translational model used for investigating the physiological impact of stress. The CUS protocol consists of unpredictable and random stressors that mimic real life exposure to stress. While stress is known to impact mitochondrial function, little is understood about the direct impact of chronic stress on renal mitochondrial respiration. Moreover, while studies have shown a link between estrogen and mitochondria through estrogen receptors, the relation between stress induced renal mitochondrial dysfunction and estrous cycle is unknown. Hypothesis: Exposing female mice to CUS will increase blood pressure and cause decreased renal mitochondrial respiration. Methods: We exposed adult female C57BL/6 mice to CUS for 28 days. Before and after stress, all control (CON, n=8) and CUS (n=10) mice were placed in metabolic cages for overnight urine collection. Urine albumin was measured by dipstick assay. On day 28, mice were assessed for blood pressure measurement via tail cuff. 24 hours later, mice were euthanized, and vaginal lavage was performed. The left kidney was removed, transected into thirds, and the poles were placed in relaxing buffer. Kidney mitochondrial oxygen consumption was assessed using high-resolution respirometry. Results: Data were stratified by phases of the estrus cycle. Neither blood pressure nor urinary albumin was significantly altered by CUS in stressed females. Females in estrus and proestrus exposed to CUS (n=5) exhibited a decrease of 20.8% ± 5.1 (n=5, p=0.03) in renal mitochondrial maximal uncoupled respiration compared to non-stressed CON. Renal mitochondrial respiration in diestrus was similar between CUS and CON. Conclusion: Results from this study reveal a link between the female estrus cycle, stress, and mitochondrial function.

This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
ETA receptor activation on vascular smooth muscle cells mediates inflammation via IL-6
Authors: Newberry Madison , Wilson Natalie , Moore Daniel , Speed Joshua
Published: May 2023

In obesity, there is a shift to a pro-inflammatory immune cell profile in adipose tissue, producing adipocyte dysfunction that leads to dyslipidemia and insulin resistance. Endothelin-1 (ET-1) is a proinflammatory peptide produced by endothelial cells that is increased in obesity. Our lab recently demonstrated that blockade of ET-1 type A receptors (ET A) attenuates the pro-inflammatory immune cell profile in adipose tissue of obese mice. This is not due to activation of ET A receptors on adipocytes because adipocyte-specific ET A knockout did not attenuate adipose tissue inflammation in obese mice. Given the abundance of ET A on vascular smooth muscle cells (VSMC), we hypothesize that ET-1 binds ET A on vascular smooth muscle cells leading to release of proinflammatory cytokines. To test this hypothesis, human vascular smooth muscle cells were treated with ET-1 and co-treated with either vehicle, BQ-123 (ET A antagonist), or BQ-788 (ET B antagonist) and mRNA and protein release of several cytokines were measured. Treatment with ET-1 led to a significant increase in interleukin-6 (IL-6) mRNA expression (77993 ± 9716 vs. 142133 ± 19181 copy count/50ng RNA) and protein release into the media (315±27 vs. 569±17 pg/mL). The increase in IL-6 production was attenuated by treatment with BQ-123 (331±7 pg/mL), but not BQ-788 (583±4 pg/mL). There was no difference in tumor necrosis factor alpha, transforming growth factor beta, or interferon gamma in cells treated with ET-1 compared to vehicle. These data suggest that elevated ET-1 may promote inflammation in obesity by increasing IL-6 production by vascular smooth muscle cells.

5R01DK124327-02

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Impaired mitochondrial quality control in skeletal muscles from C57 and D2 mdx model of Duchenne Muscular Dystrophy
Authors: Rosen Grace , Whitley Kennedy , ReyesNegron Alexander , Berger Nicolas , Lourie Jared , Cleverdon Riley , Fajardo Val , Zou Kai
Published: May 2023

Mitochondrial dysfunction is an early pathological feature of Duchenne muscular dystrophy (DMD) and precedes myopathy. Balanced mitochondrial quality control (e.g., fission, fusion, biogenesis, autophagy, and mitophagy) is essential to maintain the overall mitochondrial health and skeletal muscle function. DBA/2J mdx (D2- mdx) has emerged as a more relevant mouse model of DMD as it more closely mirrors early stage of DMD pathology in humans than the C57BL/10 mdx (C57 -mdx) model. The purpose of this study was to determine whether the expression of proteins responsible for mitochondrial quality control were dysregulated in skeletal muscles from these two mouse models of DMD. We hypothesized that the expression of protein markers of mitochondrial fission and mitophagy would be higher, while markers of mitochondrial fusion would be lower in both models of DMD with greater alterations in D2- mdx mice. 8-10-week-old male C57 -mdx and D2- mdx mice, and their respective wildtype (WT) mice were used in this study. Muscle strength was measured using hang wire test. Gastrocnemius muscles were collected for immunoblot analysis to assess mitochondrial quality control proteins. Hang wire impulse, an indicator of muscle strength, was significantly lower in both models of DMD in comparison to their WT controls (main effect of mdx, P = 0.01). Regarding mitochondrial fission, while there was no difference in protein expression and phosphorylation (Ser 616) of Dynamin-Related Protein 1 (Drp1) between any groups, mitochondrial fission 1 protein (Fis1) was markedly higher in skeletal muscles from both models of DMD when compared to WT controls (main effect of mdx, P=0.0001). Greater expression of Fis1 was observed in D2- mdx than C57- mdx when compared to their respective WT controls (~5.7-fold vs. 3.8-fold). Furthermore, mitochondrial fusion marker Mitofusion 1 (Mfn1) was significantly lower in both models of DMD (main effect of mdx, P=0.029), however, it appears to be mainly driven by the reduction in D2-mdx (mdx x strain interaction, P=0.019). No difference of Mfn1 expression was found in C57-mdx mice. Finally, expression of protein markers of mitochondrial biogenesis (PGC1 a) and autophagy (LC3B) were higher in both models of DMD when compared to their respective WT controls (main effect of mdx, P=0.0001 and P=0.001, respectively). In conclusion, these data suggest that there is dysregulated mitochondrial dynamics towards pro-fission state in skeletal muscle from mdx mice with an exacerbated effect in D2- mdx model, which may contribute to the muscle weakness phenotype associated with DMD

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Effect of 20-hydroxyecdysone on the immune response following eccentric muscle injury in C57BL/6 mice
Authors: Zwetsloot Kevin , van Onselen Lisha , Moorefield Erin , Rao Anika , Hill Levi
Published: May 2023

Previously, we demonstrated that the phytoecdysteroid, 20-hydroxyecdysone (20E), accelerates the recovery of skeletal muscle function 7 days following eccentric contraction-induced injury. We hypothesized that 20E may influence the inflammatory response during recovery from eccentric muscle injury; therefore, male mice (3-6 mo) were subjected to eccentric damage of the anterior crural muscles via 150 eccentric contractions, then treated with either 20E (50mg/kg body mass) or vehicle daily until sacrifice (12 hours, 2 days or 4 days post-injury). Using immunohistochemical techniques, infiltration of neutrophils, M1-like, and M2-like macrophages were examined in sectioned tibialis anterior muscle. Our findings revealed that pro-inflammatory responses (infiltration of neutrophils and M1-like macrophages) appear to be attenuated in 20E-treated mice at 12 hours and 2 days post-injury, respectively; whereas anti-inflammatory responses (infiltration of M2-like macrophages) appeared to increase with 20E treatment at 4 days after eccentric muscle injury. Taken together, it is possible that 20E may attenuate the overall early pro-inflammatory response, while bolstering the later anti-inflammatory response to eccentric muscle injury, leading to accelerated muscle repair/regeneration.

This research was partly funded by the Appalachian State University Office of Student Research.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Nonmuscle myosin II proteins, Myh9 and Myh10, interacts with unconventional myosin Myo18A in immortalized mouse thick ascending limb epithelial cells
Authors: Zimmerman Jenna , Busselman Brook , Otterpohl Karla , Chandrasekar Indra
Published: May 2023

Conventional nonmuscle myosin II (NM2) and unconventional myosin 18A (Myo18A) belong to an evolutionarily conserved family of molecular motor proteins that slide or walk on the actin cytoskeleton to facilitate a myriad of cellular functions including cargo transport pathways. NM2 and Myo18A are known to copolymerize into filaments in vitro in cell-free systems and in cells. Our previous work demonstrated the crucial role for Myh9&10 proteins in regulating transport of uromodulin (UMOD) and the sodium, potassium, chloride cotransporter (NKCC2) in the thick ascending limb (TAL) of mouse kidney using a Myh9&10 conditional knockout mouse model (Otterpohl, et al; 2020). Myo18A is expressed in the proximal tubule and TAL cells in the murine kidney and localizes to the apical membrane. Our results show reduced expression of Myo18A in the TAL cells of the Myh9&10 cKO mouse kidney sections. Furthermore, we identified Myo18A as a major interaction partner to the Myh10 heavy chain in a BioID screen performed in renal epithelial cells. Given that Myo18A alpha is the only myosin with a PDZ domain, we hypothesize that Myo18A coordinates with Myh9&10 in the TAL cells to regulate membrane remodeling during vesicular transport, including the targeting and stabilization of TAL proteins like UMOD and NKCC2. To test this hypothesis, we stably expressed GFP-tagged Myo18A alpha construct in immortalized, mouse thick ascending limb (imTAL) cells generated in the lab. We performed coimmunoprecipitation (Co-IP) experiments that showed interaction between Myo18A alpha, Myh9 and Myh10 proteins. Immunostaining experiments show colocalization of GFP-Myo18A alpha with Myh9 and Myh10 in imTAL cells. These results strongly support the coordinated functions of Myo18A and Myh9&10 proteins in TAL epithelia and warrants further experiments to determine their role in regulating UMOD and NKCC2 transport in the TAL cells.

1R01DK131020-01A1

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Viral vector-mediated overexpression of mitochondrial ATP-binding cassette subfamily B member7 does not protect against doxorubicin-induced cardiotoxicity
Authors: Dowllah Imtiaz Masfique , Nguyen Branden , Doerr Vivian , Jacintho Moritz Cesar , Smuder Ashley
Published: May 2023

Doxorubicin (DOX)-induced cardiotoxicity results in an increased risk of mortality among chemotherapy recipients. Cardiomyocyte dysfunction is caused by the off-target accumulation of DOX in cardiac mitochondria, stimulating oxidative damage and impaired oxidative phosphorylation. Prior research showed that exercise preconditioning may prevent DOX-induced cardiotoxicity by enhancing the abundance of mitochondrial ATP-binding cassette (ABC) transporters, potentially accelerating the rate of DOX clearance from cardiomyocytes. Mitochondrial ABC subfamily B member7 (ABCB7) has been implicated in drug export and multidrug resistance. Therefore, to further understand whether, independent of exercise, overexpression of a mitochondria-localized ABC transporter can prevent acute cardiotoxic effects of DOX, we examined the effects of adeno-associated virus (AAV)-mediated overexpression of ABCB7 on cardiac function, exercise tolerance, and mitochondrial accumulation of DOX and its metabolites (doxorubicinol) in animals treated acutely with DOX (20 mg/kg IP). Female Sprague-Dawley rats were randomized into four experimental groups (n=10/ group): 1) Saline-treatment (CON), 2) DOX-treatment (DOX), 3) ABCB7 overexpression, saline-treatment (ABCB7), and 4) ABCB7 overexpression, DOX-treatment (ABCB7+DOX). Our findings show that DOX treatment results in detriments to left ventricular fractional shortening percentage 48 hours following administration, and overexpression of ABCB7 could partially protect from such DOX-induced dysfunction. However, the effect of ABCB7 overexpression on cardiac function was not related to a reduction in mitochondrial DOX and doxorubicinol concentration, nor does it improve exercise tolerance in acute DOX-treated animals. Further work is needed to elucidate the effects of ABCB7 on mitochondrial and cardiac function.

Funding Source: R01 HL144858

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Angiotensin AT1A Receptor Signaling in AgRP Neurons in Metabolism Control
Authors: Balapattabi Kirthikaa , Ritter McKenzie , Mouradian Gary , Hodges Matthew , Sigmund Curt , Grobe Justin
Published: May 2023

Resting metabolic rate (RMR) accounts for ≈70% of energy expenditure in humans. Obesity induced by high fat diet (HFD) feeding is associated with “RMR adaptation” (i.e., reduced RMR relative to body size). We previously reported angiotensin II (ANG) receptors (AT1A) in a subset of neurons of the arcuate nucleus of the hypothalamus (ARC) that express Agouti-related peptide (AgRP) are critically required for the integrative control of RMR. Understanding signal mechanisms in this unique subset of RMR-controlling AgRP neurons is the goal of this study, and is critical to decipher the pathogenesis of obesity-associated hypertension. Cell-attached voltage clamp recording of AgRP neurons in ARC sections of Ai9AgRP mice shows that ANG application results in three distinct electrical responses in lean mice. In particular, ANG causes inhibition in ≈1/3 of AgRP neurons, and this effect is abolished by losartan or genetic deletion of AT1A, supporting involvement of AT1A receptors (“Type 1i” responses; Firing rate; aCSF:0.85 ± 0.03 vs ANG:0.20 ± 0.04 vs ANG+LOS:0.91 ± 0.11 Hz, p<0.05). After HFD-induced obesity (DIO), roughly half of the Type 1i cells begin to exhibit AT1A-dependent stimulatory instead of inhibitory responses to ANG (“Type 1s”; Firing rate; aCSF: 0.86 ± 0.03 vs ANG:1.10 ± 0.12 vs ANG+LOS:0.82 ± 0.03 Hz, p<0.05). This implies a change in AT1A second-messenger coupling within the Type 1 neurons. ANG inhibition of Type 1i cells was blocked by pre-treatment with the Gαi inhibitor, pertussis toxin (PTX) but not by Gαq inhibitor, BIM-46187 (Δ Firing rate: PTX: -0.39 ± 0.09 Hz, p<0.05 vs BIM: 0.10 ± 0.09 Hz). In contrast, ANG stimulation of Type 1s cells was blocked by BIM-46187 and not by PTX (Δ Firing rate: PTX: 0.07 ± 0.06 Hz vs BIM: 0.49 ± 0.06 Hz, p<0.05). Stereotaxic delivery of AAV constructs encoding Cre-dependent DREADDs into the ARC of AT1A-Cre mice and specific activation of hM4Di (Gαi) within AT1A-expressing (Type 1i) AgRP neurons significantly increased RMR (n= 8-10, p<0.05) in both lean and obese mice. Activation of hM3Dq (Gαq) in Type 1i AgRP neurons did not alter RMR in obese mice.These findings establish a specific ANG-inhibited subtype of AgRP neuron and implicate the AT1A/Gαi signaling cascade ultimately controls RMR. AT1A “signal switching” from Gαi to Gαq cascade within these cells may underlie the pathogenesis of RMR adaptation. These results indicate that RMR adaptation is likely secondary to the loss of AT1R—Gαi coupling, and not the gain of AT1R—Gαq coupling.

American Physiological Society and American Heart Association postdoctoral fellowship(KB) & R01 HL134850 (JLG)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Bioengineering approaches to promote maturity in human skeletal muscle constructs
Authors: Tollitt Benjamin , Jones Samantha , Henstock James , McArdle Christopher , Hoettges Kai , Jackson Malcolm , McArdle Anne
Published: May 2023

In 2021/22, the MicroAge project, a UK Space Agency-funded mission, sent 3D muscle constructs to the International Space Station to study microgravity as a model of accelerated musculoskeletal ageing on Earth. The aim of this work is to further develop the muscle constructs, compromised of human skeletal muscle cells encapsulated in fibrin hydrogels, induced to differentiate into myotubes and grown in 3D. The study uses several bioengineering approaches to determine the extent of maturity achievable in these muscle constructs in vitro, with the aim of producing a pharmacological or physiological screening platform of mature muscle, targeting muscle disorders such as sarcopenia.To enable measurement of muscle functional properties, it was also necessary to develop a suitable surrogate for force generation due to constraints of strain gauge use, and so this study also determined whether electrochemical impedance spectroscopy (EIS) was appropriate in numerous instances, opening up possibilities for rapid and automated measurement of contractile properties of in vitro skeletal muscle models. This was undertaken by the development of the MicroAge culture system to allow simultaneous measurement of force characteristic using a micro stain gauge and EIS measurements via the stimulating electrodes.The MicroAge muscle constructs have been characterised using a panel of maturation indices for skeletal muscle, including measuring contractile properties, and myosin heavy chain isoform expression using immunocytochemistry with spinning disc microscopy, RT-qPCR and Western blotting and these data will be presented.Of particular interest are the data that demonstrated a linear relationship between force generation measured using the ‘gold-standard’ approach of using a strain gauge and EIS (R2 = 0.9), validating the use of EIS as a proxy for force generation in a range of conditions such as in muscle constructs at rest and during contraction.This allows for rapid and automated measurement of contractile properties of in vitro models of skeletal muscle, providing an exciting new platform to rapidly test pharmacological interventions that may result in improved muscle function in 3D models of disease. The study is also currently implementing bioprinting of muscle constructs to streamline construct formation and investigating varying patterns of electrical stimulation on construct maturity throughout differentiation and the results of this approach will also be presented.

Supported by a PhD studentship from MRC DiMeN Doctoral Training Programme and CIMA.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Degeneration specific NF-L immunoreactivity in spinal cord and brainstem 10 days post-cervical spinal cord contusion
Authors: Fusco Anna , Rana Sabhya , Bindi Victoria , Sunshine Michael , Shaw Gerry , Fuller David
Published: May 2023

We recently described a panel of novel antibodies to epitopes on the neurofilament light chain (NF-L) which are hidden in healthy neurons and their processes but specifically exposed during neurodegeneration ( doi.org/10.1101/2022.08.27.504533 ). We saw widespread immunopositivity for such antibodies in degenerating and degenerated processes in the rat spinal cord 1-5 days following cervical spinal cord injury (cSCI). Here we aimed to determine if such immunopositivity would persist at 10 days following cSCI. Male Sprague Dawley rats received laminectomy (n=3) or right unilateral contusion at C4 (n=3; 150kdyn; Infinite Horizon Impactor). Tissue was harvested 10 days post-SCI and stained with either Cresyl Violet for routine histology or for immunohistochemistry using mouse monoclonal antibody MCA-6H63, one of several novel antibodies binding to degeneration specific NF-L epitopes (EnCor Biotechnology Inc.). Spinal cord MCA-6H63 immunopositivity was not detected in spinal cords from the laminectomy group. Contusion produced the expected histopathology with ipsilateral grey and white matter disruption and some damage to the contralateral dorsal columns. Punctate labeling for MCA-6H63 was prominent at the lesion epicenter, as well as in the ipsilateral cord rostral and caudal to the injury, consistent with axonal degeneration. Sparse, punctate MCA-6H63 positivity was observed in the contralateral cervical white matter. Qualitatively, MCA-6H63 staining was most robust in the ventral and lateral white matter ipsilateral and caudal to the lesion. This location was consistent with ongoing Wallerian degeneration in prominent spinal tracts including spinothalamic, spinocerebellar, reticulospinal, vestibulospinal and raphespinal. Bilateral MCA-6H63 staining in the dorsal columns was present mostly rostral to the injury. Some MCA-6H63 staining was noted in the medulla, most prominently ipsilateral to the lesion. We conclude that at 10 days post-cSCI this novel NF-L antibody specifically identifies degenerating and degenerated axonal profiles. This is a promising technique in assessing neurodegeneration following SCI and other neurodegenerative diseases.

R01HL139708-01A1 (DDF), R01 HL153140-01 (DDF), and EnCor Biotechnology Inc.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Physiological effects of fentanyl in goats: acute withdrawal and known and novel reversal agents
Authors: Hodges Matthew , Neumueller Suzanne , Buiter Nicole , Hilbert Grace , Pan Lawrence , Lewis Stephen , Forster Hubert
Published: May 2023

Synthetic opioids alter several vital physiological functions and discontinuation of its use can cause acute withdrawal symptoms. In this and three adjoining posters we present data on the dose-dependent physiologic and behavioral effects of intravenous (IV) fentanyl in adult awake goats. Here, we tested two hypotheses: 1) that fentanyl will lead to acute, dose-dependent withdrawal symptoms, and 2) that co-administration of a known reversal agent (naloxone (NAL)) or novel respiratory stimulant (D-cysteine ethyl ester; D-CYSee) can mitigate the acute physiological and behavioral effects of fentanyl. Acute withdrawal was measured for 4 hrs. beginning 90 min post-injection of IV vehicle or fentanyl (n=5-7) using a wearable activity tracking device (Biopac) and subjective videographic observations. Animal movement (Vector Magnitude; g) was lowest with vehicle across all time points but was increased (p<0.05) in a dose-dependent manner from 90-210 min after fentanyl administration. Additional behaviors such as pawing, vocalization, hyperphagia, itching and rearing also appeared dose-dependent and were increased from 90-210 min post-injection but waned across the 4h observation period (n=4). Based on the demonstrated efficacy of D-CYSee in reversing OIRD in rats (Getsy et al., 2022), we next tested if fentanyl-induced physiological dysfunction or withdrawal symptoms were mitigated by co-administration of either D-CYSee (500 μmol/kg) or NAL (0.06 mg/kg). Transient suppression of minute ventilation (VI) and breathing frequency was followed by a secondary increase >10 min after 50 μg/kg fentanyl, where co-administration of D-CYSee stimulated VI and NAL prevented the secondary increase in VI (n=5-6). Fentanyl alone moderately decreased HR where D-CYSee prevented this effect, whereas NAL exacerbated the fentanyl-induced bradycardia for up to 45 min. Mean, systolic and diastolic blood pressures were increased with fentanyl for >90 min post-injection, where D-CYSee had no additional effect but NAL prevented the hypertension. Finally, NAL but not D-CYSee reversed sedation (n=7), but neither NAL or D-CYSee appeared to mitigate withdrawal behaviors. These preliminary data suggest that: 1) fentanyl causes withdrawal behaviors in goats, and 2) NAL and D-CYSee have differential effects on fentanyl-induced physiological dysfunction suggesting D-CYSee has a distinctly different mechanism of action as a potential OIRD countermeasure.

Supported by NIH DA050571.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Caveolin-1 facilitates lipid uptake into endothelium by stabilizing Cd36 at the plasma membrane
Authors: Luse Melissa , Dunaway Luke , Pavelic Caitlin , Tessema Rachael , Juskiewicz Zuzanna , Minshall Richard , Leitnger Norbert , Isakson Brant
Published: May 2023

Endothelial cells (ECs) can take up lipids from the circulation through Caveolin-1 (Cav1)-mediated endocytosis. In metabolic disease, fatty acids accumulate in blood plasma, creating a pathological role for ECs as lipid metabolizing cells. ScRNAseq performed on adipose endothelium shows an inverse relationship between Cav1 expression and dietary fat percentage; with increased dietary fat consumption, there is a decrease in Cav1 mRNA. To investigate the link between EC Cav1 and lipids we employed the use of an inducible EC-specific Cav1 knockout mouse (Cav1fl/fl/Cdh5-CreERT2+). Cav1 genetic deletion from endothelium was induced at 6 weeks, and mice were fed either a normal chow (NC; 5% fat) or high fat diet (HFD; 60% fat) with all mice examined at 20 weeks. Within diet groups, all mice regardless of genotype had comparable weight gain, gonadal fat pad mass, and food consumption. Following induction, electron microscopy was used to confirm presence of caveolae in adipocytes, but not endothelium. The gross vascular network of adipose, from all experimental mice, was visualized using fluorescence microscopy following tissue clearing of whole epididymal fat pads. Deletion of EC Cav1 resulted in increased blood lipids (triglycerides, cholesterol, LDL) independent of diet, suggesting Cav1-mediated endocytosis as a major mechanism of endothelial lipid uptake under normal and pathophysiological conditions. Further investigation determined that intact endothelium of adipose arteries from mice lacking EC Cav1 had decreased cellular lipid content when compared to mice with functional Cav1. Caveolae serve as signaling microdomains in ECs, leading us to question whether the lipid uptake impairments could be due to the failure to enrich proper fatty acid uptake machinery to caveolae decreasing circulating fatty acid uptake. We found Cd36, a known fatty acid translocase, to be located within these Cav1-mediated signaling microdomains via immune staining of an adipose artery en face and cellular membrane fractionation. EC Cav1 stabilizes Cd36 at the plasma membrane and with loss of Cav1, Cd36 gets recycled into the endosomal network. The decreased localization of Cd36 at the plasma membrane impairs endothelial lipid uptake and increases plasma lipids as seen in the EC Cav1 deficient mice. This work provides initial evidence for the unique metabolic roles of endothelial

Research Support: T32 HL007284, AHA predoctoral fellowship, HL120840 (BEI), and Lipidema Foundation (BEI).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
HOX/PBX Inhibition Attenuates Tsc2-Null Cell Viability and Lung Metastasis in Lymphangioleiomyomatosis
Authors: Olatoke Tasnim , Astrinidis Aristotelis , Guo Minzhe , Zhang Erik , Zhang Alan , McCormack Francis , Xu Yan , Yu Jane , Wagner Andrew
Published: May 2023

Lymphangioleiomyomatosis (LAM) is a rare lung disease that exclusively affects premenopausal women. LAM is caused by infiltration of metastasizing atypical smooth muscle-like cells into the lungs leading to progressive respiratory failure. Though the origin of these cells remains elusive, LAM cells carry loss-of-function mutations in the Tuberous Sclerosis Complex (TSC1/TSC2) genes, leading to hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) and aberrant cell growth. There is no cure for LAM. The greatest barriers to finding a cure for LAM include its undetermined origin and unclear underlying pathogenesis. Our integrative single cell omics analyses of LAM lung identified the activation of uterine-similar HOX/PBX transcriptional network in LAM cells compared to other cells, suggesting that the uterus is the primary origin of LAM. HOX/PBX signaling is critical for female reproductive tract development and maintenance, and their overexpression is implicated in some female reproductive cancers. The objective of this study is to elucidate cellular and molecular mechanisms by which HOX/PBX signaling may regulate LAM pathogenesis, with hopes to uncover novel therapeutic strategies to cure LAM. We hypothesize that blockade of HOX/PBX attenuates LAM patient-derived (TSC2-null) cell viability and lung metastasis in LAM. We first confirmed HOX/PBX dimerization in LAM cells using immunofluorescence and immunoprecipitation assays. Using cell viability assays including crystal violet assay and MTT, we found that treatment with a cell-permeable HOX/PBX antagonist (HXR9) decreased TSC2-null cell growth and viability in vitro (*p<0.05, n=3/treatment group). We also showed that inhibiting HOX/PBX dimerization enhanced apoptosis and necroptosis in TSC2-null cells compared to controls using immunoblotting. Finally, using a xenograft mouse model of LAM, we tracked lung metastasis in vivo (Xenogen IVIS Spectrum System) and quantified total photon flux in the chest region. We found that pharmacologic and molecular suppression of HOX/PBX attenuates lung metastasis of LAM cells (*p<0.05, n=4/group). Our findings suggest that HOX/PBX signaling is critical for LAM progression and should be explored as a potential therapeutic target for LAM.

Funding Sources: Public: National Heart Lung and Blood Institute (R01HL153045) and Department of Defense (W81XWH-19-1-0474). Private: LAM Foundation (LAM0141E01-20).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Sex-specific effects of chronic unpredictable stress on mitochondrial function in the HPA axis
Authors: Crockett Alexia , Frambes Noelle , Stegmann Kyle , Churillo Amelia , Priviero Fernanda , Webb Clinton , Wood Susan , Spinale Francis , Ryan Michael , Hollis Fiona
Published: May 2023

Background: Stress is a real or perceived threat that elicits physiological and behavioral responses via activation of innate response mechanisms like the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS). Mitochondria are dynamic organelles responsible for providing cellular energy and the synthesis and release of glucocorticoids from the adrenal glands in response to stress. Thus, mitochondria represent the first line of response to environmental challenges, however, the effects of chronic stress on mitochondrial function in the HPA axis, particularly with respect to sex differences, are relatively unexplored. Hypothesis: Chronic unpredictable stress (CUS) induces mitochondrial dysfunction in key brain regions within the HPA axis.Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (CON) with weekly weighing. During the last week of stress, anxiety-like behavior was evaluated using the elevated plus maze and open field tests and coping behavior was assessed using the forced swim test. Mice were euthanized and the brain and adrenal glands dissected, weighed, and mitochondrial function was assessed using high resolution respirometry in hypothalamus, amygdala, and adrenal tissues. Results: CUS exposure decreased total body weight gain by 53% (p=0.04) and increased anxiety-like behavior (p=0.01) in stressed males compared to controls but had no effect on female weight or anxiety-like behavior. Stressed males and females both exhibited 46% decreased latency to immobility in the forced swim test (p=0.0001). CUS induced significant sex-dependent decreases in mitochondrial respiration within key stress centers, with stressed males exhibiting decreased respiration in the amygdala (20%, p=0.05), and stressed females exhibiting decreased respiration in the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06). Co nclusions: The unique findings of this study identified sex dependent CUS effects on HPA axis mitochondrial function. These observations suggest targeting mitochondria as a therapeutic strategy against stress-related pathologies.

This work was supported by a NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), & BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Deletion of the stress response protein REDD1 prevents impaired cardiac function in diabetic mice
Authors: Stevens Shaunaci , Cavus Omer , Subrahmanian Sandeep , Sunilkumar Siddharth , Toro Allyson , Pfleger Jessica , Bradley Elisa , Dennis Michael
Published: May 2023

The overwhelming impact of heart failure on morbidity and mortality in obesity and type 2 diabetes requires a deeper understanding of the molecular events that mediate cardiac dysfunction. We recently demonstrated using in vivo and in vitro models of diabetes that upregulation of endoplasmic reticulum stress in cardiomyocytes promoted expression of the stress response protein regulated in development and DNA damage 1 (REDD1). REDD1 has been implicated in diabetes-induced functional deficits in both the retina and kidney; however, a role for REDD1 in diabetes-induced cardiac dysfunction has not been investigated. Thus, we explored the hypothesis that REDD1 contributes to the development of diabetes-induced cardiac dysfunction. To induce diabetes, 6-week old male wild-type and REDD1 knock-out (KO) B6;129 mice were fed a high-fat high-sugar diet (42% kcal fat, 34% sucrose by weight) for 16 weeks; 2 weeks after initiation of the diet, mice were administered low-dose streptozotocin (STZ) for five consecutive days. Non-diabetic control mice were fed a standard rodent diet and received vehicle injections. Mice with a fasting blood glucose level >250mg/dL were defined as diabetic. At 10 weeks of age, transthoracic echocardiogram was evaluated every 2 weeks using a GE Logique small animal echocardiography machine. After 12 weeks of diabetes, left ventricular ejection fraction (EF) was reduced in diabetic wild-type mice as compared to non-diabetic wild-type mice (51.0 +/-9.8% versus 39.5 +/- 5.5%, p = 0.015). Meanwhile, EF in diabetic REDD1 KO mice (47.4 +/- 6.6%) was similar to that observed in non-diabetic REDD1 KO mice (49.6 +/- 6.7%). To investigate a potential cause of the preserved EF in diabetic REDD1 KO mice, tissue homogenates from the left ventricle were examined by PCR analysis. REDD1 mRNA expression in diabetic wild-type mice was enhanced as compared to non-diabetic controls in coordination with increased expression of Tgfb1, as well as expression of the pro-fibrotic genes Fn1, Col1a1, and Col3a1. By contrast, there was no increase in pro-fibrotic mRNA expression in cardiac tissue homogenates from diabetic REDD1 KO mice, as compared to non-diabetic REDD1 KO mice. Overall, the data support a role for REDD1 expression in the development of impaired cardiac function and the pro-fibrotic response of the heart to diabetes.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.