bluuebunny/crossref_metadata_2025 huggingface dataset:
The Sirtuin SIRT6 Represses Expression of Cachexia‐Associated Cytokine Myostatin by Blocking its NF‐kB‐Dependent Gene Transcription
Authors: Gupta Mahesh , Samant Sadhana , Bao Riyue , Pillai Vinodkumar
Published: November 2023

Introduction

Loss of body weight and involuntary muscle wasting (cachexia) is associated with many terminal diseases, including heart failure, cancer, COPD and liver cirrhosis. One of the most critical mediators of cachexia‐associated muscle wasting is myostatin (Mstn), a member of the TGFβ superfamily, which is secreted from muscle cells. Mstn binds to activin receptor‐IIB and suppresses both muscle cell growth and differentiation. SIRT6 is a chromatin associated NAD‐dependent deacetylase, which has been shown to regulate wide range of cellular processes, and considered to be an anti‐aging molecule. While SIRT6KO mice display muscle atrophy, retarded body size, metabolic defects and development of heart failure, over expression of SIRT6 has been shown to extend lifespan of mice. This study was undertaken to study the role of SIRT6 in growth and wasting of skeletal muscle.

Methods and Results

We prepared RNA from SIRT6 knocked down neonatal rat cardiac myocytes and subjected it to deep RNA sequencing. The results showed that SIRT6 deficiency led to increased expression of Mstn (22 fold) and activin receptor‐IIB (3 fold), together with decreased expression of myosin heavy chain proteins (18 fold). These results were confirmed by western blotting. We also detected significantly increased levels of Mstn in the condition media of SIRT6 knocked‐down cardiomyocytes, compared to media prepared from control cells. To test the functional significance of Mstn secreted from cardiomyocytes, we exposed C2C12 cells to media prepared from SIRT6 deficient and control cells. The condition media of SIRT6 deficient cells, but not of control cells, blocked the C2C12 cells differentiation, as measured by expression levels of myosin heavy chain protein, thus confirming that the Mstn secreted from cardiomyocytes was functionally active. To underpin the mechanism through which SIRT6 controls the expression of Mstn gene we performed the ChIP assay. We found that SIRT6 was bound to the Mstn promoter at the NF‐kB binding sites. The treatment of cells with a NF‐kB inhibitor, Bay‐11‐7082, significantly reduced the occupancy of SIRT6 to the Mstn promoter, thus suggesting that SIRT6 suppresses Mstn expression by negatively regulating NF‐kB‐dependent transcription of Mstn gene promoter.

Conclusion

Loss of SIRT6 has been linked with development of many chronic diseases. Our data suggest that induction of cachexia with these diseases may be resulting from increased expression of Mstn due to SIRT6 deficiency. Therefore, SIRT6 activation may be considered a therapeutic intervention for the management of cachexia.

Support or Funding Information

NIH RO1‐HL117041

NIH RO1‐HL111455
Identification and Characterization of Allograft Inflammatory Factor 1‐Like (Aif1L) in Skeletal Muscle
Authors: Tran Kien , Waddell David
Published: November 2023

Skeletal muscle wasting, or atrophy, frequently results from decreased protein synthesis and an associated increase in protein degradation. This shift towards a reduction in protein content of the cell can result from various physiological conditions, including immobilization, denervation, and aging. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has been found to be upregulated during almost all conditions of muscle wasting and is believed to play an important role in targeting proteins for degradation. However, preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of atrophy‐induced gene activity, including the regulation of allograft inflammatory factor 1‐like (Aif1L). Aif1L has been identified as a novel skeletal muscle gene that is upregulated in wild‐type mice, while MuRF1‐null mice show blunted upregulation in response to denervation. Aif1L is predicated to contain an EF‐hand domain, which can act as a calcium ion binding domain, and since calcium ions are crucial for proper muscle contraction this gene was investigated further. In order to confirm that Aif1L in expressed in muscle cells, the cDNA of Aif1L was cloned from the C2C12 mouse myoblast cell line and fused with a green fluorescence protein (GFP) tag. C2C12 cells were then transfected with the Aif1L‐GFP expression plasmid in order to localize the Aif1L protein in proliferating myoblast cells. C2C12 cells transiently overexpression Aif1L‐GFP showed punctate fluorescence localized to the perinuclear region of the cytoplasm. The results presented here in combination with additional research into the transcriptional regulation and function of Aif1L will help contribute new insight into the molecular genetic mechanisms of skeletal muscle atrophy and may provide a more complete understanding of how MuRF1 may transcriptionally regulate neurogenic atrophy‐induced gene expression during muscle wasting.

Support or Funding Information

University of North Florida Transformational Learning Opportunity Grant
Basal and Stretch‐induced Myotube Protein Synthesis Regulation by gp130 Signaling
Authors: GAO SONG , Carson James A.
Published: November 2023

Mechanical stimulation from muscle contractions or stretch can induce mTOR signaling and protein synthesis in cultured myotubes. Mechanical stimulation also induces muscle expression of IL‐6 family of cytokines, which can activate intracellular signaling pathways through glycoprotein 130 (gp130). Although several gp130 downstream targets, such as Akt and ERK1/2, can regulate mTOR activity, the role for gp130 initiated signaling in the stretch activation of mTOR signaling and protein synthesis has not been established. The purpose of this study was to investigate gp130 signaling on mTORC1 activation, and whether gp130 signaling is necessary for stretch‐induced protein synthesis in cultured myotubes. C2C12 myotubes were static stretched (5%) for 24h. Signaling was manipulated by gp130 siRNA transfection into cells or administration of neutralizing antibody in culture media. Phosphorylation of signaling molecules and puromycin incorporation (indicator of protein synthesis) were measured by western blot. We report that gp130 siRNA knock‐down decreased myotube basal p70S6K phosphorylation (T389) and protein synthesis. Activation of gp130 signaling by IL‐6 administration increased myotube Akt phosphorylation (T308) and protein synthesis, but not p70S6K phosphorylation. Static stretch for 24h increased myotube p70S6K phosphorylation, protein synthesis, and IL‐6 mRNA expression. Stretch in the presence of gp130 antibody attenuated the stretch‐induction of myotube p70S6K phosphorylation, and blocked the stretch‐induction of protein synthesis. These results demonstrate that gp130 dependent signaling has a role in the regulation of basal and stretch‐induced myotube protein synthesis, however, the cellular mechanism regulating protein synthesis differs between the 2 conditions.

Support or Funding Information

R01 CA121249 to JAC from the National Cancer Institute
Human Proteomes – From Basic Science To Understanding Drug Action
Authors: Kuster Bernhard
Published: November 2023

Proteomes are characterized by large protein‐abundance differences, cell‐type‐ and time‐dependent expression patterns and post‐translational modifications, all of which carry biological information that is not accessible by genomics or transcriptomics. Here we present a mass‐spectrometry‐ based draft of the human proteome and a public, high‐performance, in‐memory database for real‐time analysis of terabytes of big data, called ProteomicsDB. The information assembled from human tissues, cell lines and body fluids enabled estimation of the size of the protein‐coding genome, the analysis of messenger RNA and protein‐expression profiles of human tissues revealed conserved control of protein abundance and integration of drug‐sensitivity data enabled the identification of proteins predicting resistance or sensitivity. The same overall technology also allows tracking drug actions in cells and lysates on a target‐class or proteome‐wide scale with many implications for drug discovery. We present examples for the use of chemical probes and chemical proteomics to find new uses of existing drugs, to identify toxicity targets, to elucidate the mechanism of action of drugs in cancer cells, the identification of resistance mechanisms and how the activity status of cancer cells as assessed by post‐translational modification of proteomes influences the efficacy of drugs.

Support or Funding Information

Center for Integrated Protein Science Munich

German Consortium for Translational Cancer Research

SAP SE
Expression of the Dystrophin‐Associated Protein Complex in the Absence of Skeletal Muscle AMPK
Authors: Dial Athan G , Lally James S , Bujak Adam L , Steinberg Gregory R , Ljubicic Vladimir
Published: November 2023

The dystrophin‐associated protein complex (DAPC) is composed of proteins that are highly expressed along the sarcolemma. The DAPC provides a mechanical link between the intracellular cytoskeleton and extracellular matrix, as well as a signal transduction apparatus from the periphery to the interior of muscle fibers. The signalling molecule AMP‐activated protein kinase (AMPK) is a powerful regulator of phenotypic plasticity. Recent evidence has shown that chronic AMPK activation alters the expression of DAPC components. However, a more comprehensive understanding of the influence of AMPK on the DAPC is lacking. Therefore, the purpose of this study was to investigate the role of AMPK in the expression of the DAPC. Extensor digitorum longus (EDL) and soleus (SOL) muscles representing fast glycolytic and slow oxidative tissues, respectively, were obtained from wild‐type (WT) mice, as well as from mice deficient in both isoforms of the AMPK‐β subunit in skeletal muscle (AMPK‐MKO). RT‐PCR and Western blotting measured mRNA and protein content of DAPC components, respectively. In WT animals, utrophin and laminin mRNA expression, as well as laminin and β‐dystroglycan protein content were ~25–60% higher (p < 0.05) in the SOL versus EDL muscle. In contrast, neuronal nitric oxide synthase (nNOS) mRNA was 60% lower in SOL. The AMPK‐MKO animals displayed a different pattern of fiber‐type specific expression of DAPC components. In these mice, laminin, γ‐sarcoglycan (SG), dystrophin, and utrophin transcripts, as well as dystrophin and utrophin protein expression were 40–55% greater (p < 0.05) in the SOL versus the EDL. β‐SG and nNOS mRNA content were lower in the EDL muscle of AMPK‐MKO mice, as compared to their WT counterparts. We also assessed the expression of peroxisome proliferator activated receptor coactivator‐1α (PGC‐1α) and Ca2+/calmodulin dependent protein kinase II (CAMKII), factors that contribute to the upstream regulation of the DAPC. PGC‐1α mRNA content was higher in SOL relative to EDL muscles of both genotypes. CAMKIIα protein expression was significantly lower in SOL muscles versus EDL muscles of WT and AMPK‐MKO, while CAMKIIβ exhibited the opposite pattern. PGC‐1α mRNA tended to be higher in the muscles from AMPK‐MKO mice compared to WT animals. Conversely, CAMKII mRNA content tended to be reduced in the AMPK‐MKO muscles, as compared to WT. These data indicate a fiber‐type specificity to DAPC expression in skeletal muscle. Furthermore, AMPK deficiency results in a differential profile of DAPC components, as well as in alternations in the expression of alternative upstream DAPC regulators. Our results suggest that AMPK contributes to the expression profile of the DAPC.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada, Canada Research Chairs
Regional Gradients in Tissue Anisotropy During Pressure Overload Hypertrophy in the Rat Left Ventricle
Authors: Carruth Eric D. , McCulloch Andrew D. , Omens Jeffrey H.
Published: November 2023

Many studies have shown that the structural composition of the normal heart is spatially non‐uniform. We hypothesized that due to spatial differences in tissue structure and function, hypertrophic growth occurs non‐uniformly, especially in moderate hypertrophy. Using high resolution, high fidelity diffusion tensor magnetic resonance imaging (DT‐MRI), we calculated and compared fractional anisotropy (FA) in several regions of aortic banded and sham hearts.

Preliminary results indicate that the FA is decreased uniformly in severe hypertrophy (mean 0.16) compared to sham controls (mean 0.22). A decrease in FA is consistent with known changes in myocyte shape during concentric hypertrophy, wherein cell width increases more than cell length. In mild‐moderate hypertrophy, the mean FA across the entire LV (0.21) was comprised of two subpopulations with mean FA 0.11 and 0.23. The subpopulation with lower FA was isolated to the subendocardium, whereas the higher FA values were localized in the mid‐and subepicardium. We conclude that hypertrophic growth occurs either earlier, with higher sensitivity, or both in the subendocardium, and we propose that endogenous spatial differences in cell and tissue structure (and therefore function) lead to this variation in hypertrophic growth.

Support or Funding Information

NIH/NHLBI Training Grant #2015‐0453NIH/NIGMS #P50 GM094503‐04
A Role for the Transient Receptor Potential Vanilloid 4 Channel in Modulating Uterine Tone During Pregnancy
Authors: Ying Lihua , Alvira Cristina M. , Cornfield David N.
Published: November 2023

BACKGROUND

The mechanisms responsible for the sustained myometrial smooth muscle cell (mSMC) contraction in active labor are incompletely understood. Transient receptor potential vanilloid (TRPV) channels are a novel class of non‐voltage gated membrane cation channels activated by swelling, stretch, and osmotic stimuli. Previously, we demonstrated that TRPV4 channel expression in mSMC increases with gestation in rats and mouse and that TRPV4 protein expression increases disproportionately more in the membrane than cytosolic fraction of mSMC.

OBJECTIVE

We hypotheses that TRPV4 channel plays a role in oxytocin induced increases in mSMC cytosolic calcium and modulates uterine contractility.

DESIGN/METHODS

Uterine muscle strips were isolated and stimulated with oxytocin, in the presence and absence of the TRPV4 channel antagonist HC 067047 and measured the contraction responses. Control wild‐type (WT) C57/B6, TRPV4 knockout (TRPV4−/−) mice were used. Contraction tracings were recorded using the POLYVIEW. The area under the curve was calculated for the final 3 minutes of each 9‐minute contraction tracing using the LabChart Reader Software. The area under the curve for each response was then normalized to the area under the curve for the spontaneous contraction pattern over the 5‐minutes period that immediately preceded the challenge.

RESULTS

To test the hypothesis that TRPV4 channel activity modulates the uterine contractile response to oxytocin, we studied the contractile response of uterine muscle strips from pregnant (day 18) WT mice exposed to increasing concentrations of oxytocin (1–1000 nM) from 1.08fold±0.96 to 3.30fold±0.39 (Area under curve, mean±SE) in the presence and absence of a specific TRPV4 channel antagonist HC 067047 (from 1.05fold±0.66 to 1.88fold±0.33), as well as the contractile response to the TRPV4 agonist, GSK1016790A. To further clarify the role of the TRPV4 channel in uterine contractility, we measured the contractile response of uterine strips derived from pregnant (day 18) TRPV4 null mice to oxytocin. Uterine strips from WT mice demonstrated a dose‐dependent contractile response to oxytocin that was attenuated by pharmacologic blockade of the TRPV4 channel. The TRPV4 channel agonist caused a dose‐dependent increase in uterine contractility. Uterine strips from TRPV4 null mice were less responsive to oxytocin than uterine strips from WT mice.

CONCLUSIONS

TRPV4 channel activity or expression increase oxytocin‐induced myometrial strips contraction.
Myostatin Protein and Muscle Fiber Cross‐Sectional Area are Associated with Increased Muscle Satellite Cell Number in 10 Month Old Fisher 344 Rats Treated with Testosterone and Trenbolone Enanthate
Authors: Beck Darren T. , Dalbo Vincent J. , Mobley C. Brooks , Ballmann Christopher , Kephart Wesley C. , Fox Carlton D. , Santucci Vincent A. , Beggs Luke A. , Balaez Alexander , Hoerr Frederic J. , Yarrow Joshua F. , Borst Stephen E. , Roberts Michael D.
Published: November 2023

Influence of 5α‐reductase and/or aromatase enzymes on androgen‐induced skeletal muscle maintenance in older adults is not fully elucidated. Our purpose was to determine the androgen‐induced alterations in adult rodent skeletal muscle fiber cross sectional area (fCSA), satellite cell content, and myostatin (Mstn) signaling by comparing the effects of testosterone (TEST) versus trenbolone (TREN, a non‐5α‐reducible, non‐aromatizable synthetic TEST analogue) in 10 month old Fisher 344 rats (n=41) randomly assigned to receive Sham surgery, orchiectomy (ORX), ORX+TEST (7.0 mg/week), or ORX+TREN (1.0 mg/week). After 29 days animals were euthanized and the levator ani/bulbocavernosus (LABC) muscle complex was weighed and preserved for analyses. LABC muscle fCSA after ORX was 37% lower than SHAM and ~50% lower than ORX+TEST and ORX+TREN (p<0.01). LABC fCSA was 28% higher in ORX+TEST compared to SHAM (p=0.012). ORX+TEST and ORX+TREN increased satellite cell numbers compared to SHAM and ORX (p<0.05), with no differences between conditions for myonuclear number per muscle fiber (p=0.948). Mstn protein was increased roughly 3‐fold in the ORX+TEST and ORX+TREN groups versus Sham and ORX groups (p<0.01). Mstn mRNA, was lower in ORX, ORX+TEST and ORX+TREN compared to SHAM (p<0.05). Muscle fCSA was highly positively correlated with muscle satellite cell number (r = 0.730, p < 0.001). Mature Mstn protein was positively correlated with muscle satellite cell number (r = 0.430, p = 0.008). TEST and TREN increased muscle fCSA and satellite cell number without increasing myonuclei number, and increased Mstn protein levels. Mstn protein and Muscle fCSA were associated with increased muscle satellite cell number. TEST and TREN appear to up‐regulate Mstn protein levels to prevent uncontrolled muscle growth in older animals. In addition, the similar effects of TEST and TREN observed in the current study suggest the action of 5α‐reductase and/or aromatase enzymes are inconsequential for the androgenic maintenance of skeletal muscle in older adult males.

Support or Funding Information

LABC analyses were performed at Auburn University and reagents for this study were purchased by discretionary laboratory funds from M.D.R. This study was also supported in part by work supported from the Office of Research and Development, Rehabilitation Research and Development (RR&D) Service, Department of Veterans Affairs Merit Award to S.E.B., Veteran's Health Administration Advanced Geriatrics Fellowship to D.T.B., and RR&D CDA‐2 to J.F.Y.
An investigation of the suitability of using octanoylcarnitine together with malate as a substrate combination for assessing Beta‐oxidation using High Resolution Respirometry
Authors: Osiki Prisca Ofure , Ojuka Edward Otim , Marmaan Gerald
Published: November 2023

Background

Beta‐oxidation is often measured using respirometry with octanoylcarnitine + malate as substrates in cells. Malate is necessary to ensure continuous oxidation of octanoylcarnitine. However, since malate is metabolized in the TCA cycle, it is not clear if its inclusion as a co‐substrate allows for a valid assessment of beta‐oxidation when TCA cycle function is compromised.

Aim

To investigate the validity of beta‐oxidation assessment using octanoylcarnitine + malate as a substrate combination in skeletal muscle when mitochondrial (mt) aconitase is inhibited.

Methods

Soleus muscle fibres (1.5–2mg) from healthy male Wistar rats were permeabilized with saponin and incubated for 45 minutes with 1mM oxalomalic acid (aconitase inhibitor) or 1mM 2‐mercaptoacetate, an inhibitor of MCAD – the rate‐limiting enzyme of octanoylcarnitine oxidation. Respiration at Leak, Oxphos and ETS states were measured using an Oroboros oxygraph. Citrate and 2‐oxoglutarate in the respiratory media were measured using CG‐MS. Activities of aconitase and MCAD were determined spectrophotometrically.

Results

Oxalomalic acid (1mM) and 1mM of 2‐mercaptoacetate caused 24% and 58% inhibition of acnonitase and MCAD, respectively. Oxygen flux at Oxphos (0.5 ± 0.3 pmol.S−1.mg−1) and ETS (0.6 ± 0.2 pmol.S−1.mg−1) decreased in 2‐mercaptoacetate‐treated samples by 62.5% and 60%, respectively, but were unchanged with oxalomalic acid treatment. Respiration at leak state was similar for all treatments. Citrate level in the medium increased by 2‐fold at Oxphos state after 30 minutes.

Conclusion

Octanoylcarnitine + malate allows for a valid assessment of beta‐oxidation capacity using respirometry under conditions where mt‐aconitase has been inhibited.

Support or Funding Information

Support: 1. The research unit for Exercise Science & Sports Medicine at the University of Cape Town 2. The National Research Foundation (NRF), South Africa, for funding the research.
Cardiac Aging Concurs With Abnormal Mitochondrial Metabolism, Calcium Handling and Disrupted Levels of Mitochondrial Calcium Uniporter Complex Member Proteins
Authors: Suarez Jorge , Diaz‐Juarez Julieta , Scott Brian T. , Dai Anzhi , Dillmann Wolfgang H.
Published: November 2023

Cardiac aging results in impaired cardiac function along with mitochondrial (Mito) dysfunction. The mechanisms for diminished function of the aging heart are incompletely investigated. Mito metabolism is regulated by free calcium concentration ([Ca2+]m) in the mitochondrial matrix. The Mito calcium uniporter complex (MCUC) is the main calcium transporter in the mitochondria. We hypothesized that Mito dysfunction could be the result of aging‐induced maladaptive changes in protein levels of the MCUC that results in deficient Mito Ca2+ handling that, in turn, impairs Mito metabolism. Therefore, the objective of this work was to determine the protein levels of members of the MCUC and analyze Mito Ca2+ handling as well as glucose oxidation (Gox). We measured protein levels by Western blot of MCU, MCUb, EMRE and MICU1/2 in hearts of young (2–3 months) and old (19–22 months) mice. In addition, we analyzed Mito Ca2+ uptake, release, and [Ca2+]m using Mito pericam. Gox was measured in an isolated “working heart” preparation. Our results showed that MCU and EMRE, which stimulate Ca2+ import, are decreased by 22% and 83% in hearts from old mice, respectively. In addition, MCUb which inhibits Ca2+ import is increased by 65% with no changes in MICU1/2. Furthermore, Mito Ca2+ uptake and [Ca2+]m were decreased by 35% and 37%, respectively. These effects of aging on Mito Ca2+ handling were consistent with reduced Gox by 45% that we measured in hearts from old mice.

In conclusion, aging is associated with abnormal Mito Ca2+ handling produced by decrease in proteins of the MCUC that stimulate Mito Ca2+ uptake and increase in expression of MCUb, which inhibits Mito Ca2+ uptake. These maladaptive changes in aging result in impaired Mito metabolism and reduced Mito function in the heart.

Support or Funding Information

This work was supported by National Institutes of Health grants (5 P01 HL066941‐13), with a Merit Review Award from the Department of Veteran's Affairs (5 I01BX001121‐02) and the P. Robert Majumder Charitable Foundation. Diaz‐Juarez J. received support from UCMEXUS‐CONACYT.
The Role of Mitophagy in Doxorubicin‐Induced Cardiomyocyte Death
Authors: Weiner Ashley , Kaminaris Amanda , Kobayashi Satoru , Gerdes Martin , Liang Qiangrong
Published: November 2023

Doxorubicin (DOX), one of the most commonly used antineoplastic agents, is effective against many cancers, but limited by its toxicities on non‐cancerous cells, especially cardiomyocytes. Although some strategies have been attempted, development of a specific therapy targeting DOX‐induced heart failure has not yet been created, largely because the mechanism remains poorly understood. DOX sets off a cellular cascade leading to mitochondrial disruption, with cell death being the ultimate event. Since mitochondrial density is up to 35% in cardiomyocytes, DOX‐induced mitochondrial malfunction can cause a serious shortage of energy supply. In this study, we investigated whether DOX treatment results in excessive elimination of mitochondria through the autophagy‐lysosome pathway known as mitophagy, and if this leads to an increase in cardiomyocyte death. Mitochondrial morphology and the levels of mitophagy in DOX‐treated H9c2 cardiac myoblasts were examined with adenovirus‐encoded mtRosella, a mitochondrial‐targeted pH sensitive dual fluorescent fusion protein. Confocal imaging analyses show that DOX enhanced mitochondrial fragmentation and increased mitophagy. To determine the underlying mechanism that mediated DOX‐induced mitophagy, we examined the expression levels of several proteins that regulate mitophagy. The serine/threonine protein kinase PINK1 and the E3 ubiquitin ligase Parkin comprise a well‐established pathway involved in the activation of mitophagy. The protein levels and mitochondrial translocation of Parkin and PINK1were all increased at 2 hours after DOX treatment, but only the effects on PINK1 were sustained at 24 hours. Parkin levels were actually reduced at this time point. These results suggest that the PINK1/Parkin pathway may contribute to DOX‐induced mitophagy and cell death. Indeed, overexpression of Parkin via adenovirus‐mediated gene transfer accelerated DOX‐induced mitochondrial morphology changes and induced mitophagy, which was associated with exaggerated cell death as indicated by propidium iodide (PI)‐uptake and cellular levels of cleaved caspase‐3 and cleaved Poly ADP ribose polymerase. Conversely, Parkin knockdown by siRNA inhibited DOX‐induced mitophagy and reduced apoptotic marker levels and PI‐uptake. These results suggest that DOX‐induced mitochondrial fission and mitophagy are detrimental to cardiac cells. Strategies that limit mitochondrial degradation through PINK1/Parkin mediated mitophagy may help reduce DOX cardiotoxicity.

Support or Funding Information

This study is supported by the following NIH grant: 1R15HL120027‐01A1
The Requirement of Cytochrome c Oxidase for Mitochondrial Fusion in Copper‐induced Regression of Cardiomyocyte Hypertrophy
Authors: Yin Wen , Li Rui , Feng Xiaorong , Kang Y. James
Published: November 2023

Copper (Cu) supplementation reverses cardiomyocyte hypertrophy along with recovery of cytochrome c oxidase (CCO) activity and morphology of mitochondria. The present study was undertaken to test the hypothesis that Cu induces recovery of mitochondrial morphology by regulating CCO activity in hypertrophic cardiomyocytes. Primary cultures of neonatal rat cardiomyocytes were exposed to phenylephrine (PE) at a final concentration of 100 mM in cultures for 48 hrs to induce cell hypertrophy. Depressed CCO activity and fragmentation of mitochondria were observed in the hypertrophic cardiomyocytes. Cu addition to the cultures of hypertrophic cardiomyocytes at a final concentration of 5 mM for 24 hrs recovered CCO activity and led to mitochondrial fusion, along with regression of cell hypertrophy. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a Cu chaperone for CCO, led to fragmentation of mitochondria. The COX17 deficiency‐mediated CCO depression suppressed Cu‐induced mitochondrial fusion, along with an irreversibility of cell hypertrophy. This study thus demonstrates that CCO is required for mitochondrial fusion, which is critically involved in the Cu‐induced regression of cardiomyocyte hypertrophy.

Support or Funding Information

Supported by National Science Foundation of China (NSFC grant 81230004).
A High Methionine, Low Folate and Low Vitamin B6/B12 Diet Causes Neurovascular Dysfunction and Subsequent Short‐Term Memory Loss
Authors: Nuru Mohammed , Kalani Anuradha , Muradashvili Nino , Lominadze David , Tyagi Neetu
Published: November 2023

Methionine is an amino acid found in rich quantities in proteinaceous foods such as meats, fish and eggs. This essential amino acid is high in the average American diet due to protein consumption that far exceeds the daily requirement. Besides previous studies showing a correlation between high methionine and osteoporosis, heart attacks and pregnancy complications, more recent studies are showing links between high methionine and neurodegenerative disorders such as Alzheimer's and dementia. However, the mechanisms of methionine's effects are not clear. Therefore, this study is focused on the effects of a high methionine diet on neuronal and vascular function in the brain. 8–10 week old C57BL6/J male mice were fed with a methionine enriched (1.2%), low folate (0.08mg/Kg), and low vitamin B6 (0.01mg/Kg) and B12 (10.4 μg/Kg) diet for a 6 week period. Neuronal density and their degeneration were evaluated by assessing levels of Neuronal nuclear antigen and Neuron‐specific enolase (NSE), while vascular permeability was evaluated by assessing levels of endothelial junction proteins Vascular Endothelial‐Cadherin (VE‐Cadherin) and Claudin‐5 in harvested brain tissue. Cerebrovascular permeability was assessed by evaluating microvascular leakage of fluorescently‐labeled albumin in vivo. Cognitive function of mice was determined by measuring short‐term memory using a Novel Object Recognition test (NORT). Western blot and Immuno‐histochemical analyses of neuronal‐degeneration and vascular junction markers revealed a weekly increase in neuronal and vascular damage in mice on the diet. A significant increase in neuronal damage (Integrated optical density (IOD) of NSE/GAPDH: 1.39 +/− 0.01) was found in mice on the diet for 4 weeks compared to that (IOD of NSE/GAPDH: 0.99 +/−0.02) in control animals. Vascular albumin leakage was increased and VE‐cadherin levels (IOD of VE‐cadherin/GAPDH: 0.28 +/− 0.05) was decreased in mice on diet for 6 weeks compared to vascular leakage and VE‐cadherin levels (IOD of VE‐cadherin/GAPDH: 0.48 +/− 0.02) in control mice. NORT showed a decrease in short‐term memory (discrimination index (DI): 0.45 +/− 0.15) in mice on diet for 4 weeks compared to the wild‐type control group (DI: 0.89 +/−0.13). Collectively, our results suggest that a diet containing high methionine, low folate and low vitamin B6/B12 results in increased neuronal degeneration and vascular dysfunction, leading to short‐term memory loss. Interestingly, significant neuronal damage precedes vascular dysfunction.

Support or Funding Information

This work was supported in part by NIH grants HL107640‐NT, NS084823 and P30 GM103507.
Hypolipidemic effects of tomato in rats fed a high‐fat diet
Authors: del Carmen Alfaro ‐ Olivera María , Calla‐Pampa Mitchell Ricky , Fernández‐Rodríguez Lissett Jeanette
Published: November 2023

Hyperlipidemia is a pathological condition that contributes to the development of cardiovascular diseases such as atherosclerosis. Different studies have shown that levels of triglycerides, cholesterol, HDL and LDL were modified by eating tomato. The aim of this study was to determinate the effects of tomato in rats fed a 20% fat diet including palm oil for 2 weeks. The rats were then randomly divided into control and treatment groups. Tomato (0.017 kg/day) was administered to the treatment group for 30 days. Total cholesterol, triglycerides, HDL and LDL were measured in both groups. The results demonstrate that tomato reduced the concentration of plasma lipids in the treatment group (cholesterol, 99.7 mg/dl; triglycerides, 67.8 mg/dl); compared to control group (cholesterol, 131.3 mg/dl; triglycerides, 118. 7 mg/dl ; p <0.01). In conclusion, tomato consumption has a statistically significant hypolipidemic effect in rats.
Signaling in the Catalytic Subunit of Protein Kinase A via Hydrophobic Motifs
Authors: Veglia Gianluigi
Published: November 2023

Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate several signaling events. Protein kinase A is a prototypical kinase of paramount biological importance as it is involved in a myriad of cellular processes. Using side‐chain methyl group NMR relaxation measurements, we traced the allosteric signaling through the hydrophobic spines of the enzyme in the apo, binary (nucleotide bound) complex and ternary (nucleotide and pseudo‐substrate bound) forms. In the apo form, the C‐spine is disassembled, with the two lobes of the enzyme dynamically uncommitted. Nucleotide binding locks the architecture of the catalytic spine, synchronizing the motions (committed dynamics) in the hydrophobic core. While pseudo‐substrate binding further rigidifies of the spines, the conformational dynamics of the core are retained with natural substrates. Since the C‐subunit is highly conserved within the kinase family, the present study offers novel mechanistic insights into intramolecular signaling of protein kinases that can serve for the design of novel activators or inhibitors of kinases.
Heightened Anaphylaxis Reaction in Female Mice is Associated with Increased Synthesis of Mast Cell Secretory Granule‐Associated Immune Mediators
Authors: Mackey Emily , D'Costa Susan , Pohl Calvin , Ayyadurai Saravanan , Laster Scott , Moeser Adam James
Published: November 2023

Sex is a critical determinant in the prevalence and severity of many diseases. In particular mast cell (MC)‐associated diseases, including irritable bowel syndrome (IBS) and IgE‐mediated allergy, are more prevalent in females. Our previous studies with murine and porcine allergy and stress models demonstrated that females exhibit more severe anaphylaxis and MC‐mediated intestinal permeability. We hypothesized that heightened disease responses in females are associated with sex‐specific differences in MC reactivity. Our objective was to determine whether MCs exhibit sex‐specific differences. Utilizing bone marrow‐derived mast cells (BMMCs) derived from female and male mice, we showed that female BMMCs exhibited greater mediator release than male BMMCs in response MC stimuli. Further experiments revealed that heightened MC granule mediator release in females was not due to increased MC degranulation signaling, but a result of female MCs increased capacity to store granule mediators. To investigate a mechanism for increased mediator content in female MCs, we performed transcriptional analysis of male and female BMMCs. Results from sequencing revealed that female MCs demonstrated higher expression of genes related to mediator synthesis and secretory granule maturation processes. Taken together, these data revealed that female MCs have increased storage of mediators, likely due to the higher expression of genes related to mediator synthesis and granule maturation factors. The findings are in line with the heightened anaphylactic response found in female mice. Uncovering the mechanism responsible for sex‐dependent differences in MCs could allow for the development of sex‐specific and more effective treatments for MC‐associated diseases.

Support or Funding Information

This research was supported, in part, by the following grants from the National Institutes of Health: R01 HD072968 (AJM), R03 DK097462 (AJM), and T35 OD011070 (EM)
17β‐Estradiol and Bisphenol‐A Exacerbate Symptoms of Dextran Sodium Sulfate Induced Colitis in Mice
Authors: DeLuca Jennifer A.A. , Allred Kimberly F. , Jayaraman Arul , Allred Clinton D.
Published: November 2023

Inflammatory bowel diseases (IBDs) are a collection of chronic disorders associated with inflammation of gastrointestinal (GI) epithelium and autoimmune responses in the GI tract. Crohn's disease (CD) and ulcerative colitis (UC), the two main IBDs, have differing responses to increased concentrations of estrogen in women suffering from these conditions. Evidence suggests that higher estrogen exposure, such as that associated with hormone replacement therapy or normal fluctuations during menses, are related to symptom reduction in women with CD but exacerbation of symptoms in those with UC. In the Dextran Sodium Sulfate (DSS) induced model of colitis, co‐administration of estrogen has previously shown negative effects on disease activity scores in mice. The effects of xenoestrogens on DSS‐induced colitis have not been evaluated. One such xenoestrogen, Bisphenol‐A (BPA), is a known endocrine disruptor with estrogenic effects mainly via estrogen receptor β, the dominant estrogen receptor in the GI tract. In this study, we investigated the ability of 17β‐estradiol (E2) and BPA, to exacerbate colonic inflammation due to acute DSS‐induced colitis in vivo. Co‐treatment of DSS and E2 resulted in 100% mortality, with initial deaths observed 72 hours after the start of DSS treatment. DSS and BPA together resulted in 67% mortality, with most deaths between 120 and 168 hours after initial DSS treatment. Body weight, an overall indicator of disease severity, decreased significantly in E2 mice. BPA treatment also decreased body weight, with initial decrease occurring later than that observed in E2 mice. Rectal bleeding scores were most severe in E2 mice, with the greatest increase in severity between 48 and 72 hours following initiation of DSS. Between 48 and 120 hours after DSS treatment began, rectal bleeding scores of BPA mice worsened and were intermediate between E2 and control mice. Fecal consistency scores were most severe in E2 mice. Fecal scores tended to be worse in BPA mice compared to controls, but this was not significant. DSS treatment significantly decreased colon length, as expected. Ongoing analysis focuses on evaluation of cytokine expression and pathological examination of tissues. This is the first study to demonstrate that a xenoestrogen introduced through the diet can exacerbate symptoms of UC and suggests that investigating how exposure to BPA might influence UC in humans is warranted.

Support or Funding Information

Funding was provided by the Center for Translational Environmental Health Research through National Institute of Environmental Health Sciences grant number P30ES023512.
Regulation of the Antioxidant Glutathione by Enteric Glia During Inflammation
Authors: Brown Isola A.M. , Gulbransen Brian D.
Published: November 2023

Enteric glia are a unique type of peripheral neuroglia that surround and support neurons in the enteric nervous system. Enteric glia serve a dichotomous protective/pathogenic role where activated glia contribute to neurodegeration via mechanisms involving oxidative stress (Brown et. al, 2015). Conversely, the ablation of glia leads to neurodegeneration and altered gastrointestinal (GI) motility patterns (Aubé et. al, 2006; Abdo et. al, 2010). This suggests that a loss of glial neuroprotective factors such as reduced glutathione (GSH) may also contribute to neurodegeneration in disease. Here, we investigate the role of glial GSH in GI pathology. Specifically, we tested the hypotheses that glial GSH content is dysregulated during GI inflammation and that inhibition of glial GSH production can contribute to neurodegeneration.

We measured neuronal and glial content of reduced/oxidized glutathione (GSH/GSSG) using antibodies against glutaradehyde‐linked and free GSH/GSSG. We measured neuronal survival by immunohistochemistry and inhibited GSH synthesis using Buthionine Sulfoximine (BSO), an inhibitor of the GSH synthesis enzyme glutamyl‐cysteine ligase. We induced inflammation in vivo using 2,4‐dinitrobenzene sulfonic acid (DNBS) and in situ using the P2X7 receptor (P2X7R) agonist BzATP. All experiments used male C57Bl/6 mice, aged 8–10 weeks. Data was analyzed using a Student's t‐test or one‐way ANOVA as appropriate.

Total glial glutaradehyde‐linked GSH/GSSG content was not altered following in vivo DNBS colitis (p= 0.81). Free neuronal oxidized glutathione (GSSG) was increased during DNBS colitis (p<0.001) with no corresponding change in neuronal reduced glutathione (GSH), thus increasing the ratio of oxidized/reduced glutathione (GSSG/GSH) (p<0.05). Surprisingly, the ratio of neuronal oxidized/reduced glutathione did not increase during an in situ model of neuroinflammation with the P2X7R agonist BzATP. In situ inhibition of endogenous GSH synthesis decreased myenteric plexus neuronal density (p<0.01) but did not potentiate P2X7R mediated neuron loss.

Our data suggests a role for glial GSH where glia produce neuroprotective GSH in response to increased neuronal oxidative stress during GI pathophysiology. Inhibition of this protective capacity of glia with BSO makes enteric neurons more susceptible to neurodegeneration and may be a potential mechanism explaining the susceptibility of specific neuronal populations to oxidative stress and neurodegeneration during inflammation.

Support or Funding Information

NIEHS Training Grant in Environmental Toxicology to I.B. and Crohn's and Colitis Foundation of America Senior Research Award to B.G.
Bile acids regulate intestinal epithelial restitution: implications for pathogenesis and therapy of IBD
Authors: Lajczak Natalia Katarzyna , Mroz Magdalena Slawa , Saint‐Criq Vinciane , Keely Stephen
Published: November 2023

Epithelial restitution is an essential process for maintenance of intestinal barrier function. Increased levels of colonic bile acids have been proposed to be involved in the pathogenesis of inflammatory bowel disease (IBD) but their roles in regulating restitution are not yet known. Here, we investigated the effects of bile acids on epithelial restitution and molecular pathways involved in colonic epithelial healing.

T84 colonic epithelial cells, grown as monolayers on transparent permeable supports, were wounded by scratching with a pipette tip at T=0. Cells were treated with either the most abundant colonic bile acid, deoxycholic acid (DCA; 150 μM), the “therapeutic” bile acid, ursodeoxycholic acid (UDCA; 100 μM), a Farnesoid X Receptor agonist, GW4064 (5 μM), or a cystic fibrosis transmembrane conductance regulator (CFTR) channel blocker, CFTR(inh)‐172 (10 μM). Restitution was measured as wound area after 48 h expressed as % T=0 wound area. HEK‐293 cells were transfected with vector expressing luciferase gene under control of the CFTR promoter and vectors expressing FXR. Protein expression was assessed by western blotting and cell migration by Boyden chamber assay.

After 48 h post‐wounding, wound closure in untreated cells was 63.3 ± 13.5% of that at T=0, while in cells treated with DCA (150 μM) it was reduced to 24.5 ± 13.1 % (n = 5; p <0.001), whereas UDCA enhanced healing to 88 ± 4 (n = 5; p < 0.001). Furthermore, UDCA prevented inhibition of wound closure by DCA. The effects of DCA are mediated via a decrease in cell migration to 0.7 ± 0.1 fold (n = 5, p < 0.05) of that in untreated controls, rather than inhibition of cell growth. Furthermore, DCA decreased cell surface CFTR expression to 23 ± 5% of controls (n = 3, p < 0.001), while a CFTR inhibitor, CFTR(inh)‐172 (10 μM), attenuated wound closure to 37 ± 2 % (n = 5; p < 0.01), compared to. Moreover, DCA decreased CFTR promoter activity, in a concentration‐dependent manner that was also dependent on co‐expression of FXR. Finally, GW4064 (5 μM), an agonist of FXR, mimicked DCA effects on wound healing and CFTR expression.

Our data suggest that colonic bile acids differentially regulate intestinal epithelial restitution and that UDCA promotes healing and protects against the detrimental effects of DCA. Thus, manipulation of the colonic bile acid pool may prove to be a useful approach for promoting intestinal barrier function in IBD.
IBS‐induced constipation alters gut microbiota stability leading to intestinal transit time changes in the host
Authors: Touw Ketrija , Nadimpalli Anuradha , Hubert Nathaniel , Leone Vanessa , Kashyap Purna , Chang Eugene
Published: November 2023

Pathogenesis of Irritable Bowel Syndrome (IBS) is multifactorial, and gut microbes are sought to play a role in development and progression of disease. In this study we examined how constipation‐predominant Irritable Bowel Syndrome (IBS‐C) microbiota affect host intestinal transit and signaling pathways. To determine IBS‐C dysbiosis effects on the host, we introduced fecal microbiota from constipated mice to germ‐free recipients. Recipient mice of IBS‐C microbiota developed delayed GI transit time when compared to controls. Bacterial DNA analysis by HiSeq sequencing showed significant increase in Bacteroides uniformis, Bacteroides ovatus and Parabacteroides distasonis species from Bacteroidetes phylum in constipated mice. Fecal microbiota metabolic potential was measured with tetrazolium dye assay and showed significant differences between recipients of control and IBS‐C microbiota. Microbial metabolites such as short chain fatty acids (SCFA) are known to alter intestinal transit. SCFA levels in cecal contents of recipeints were measured by GC‐MS, and showed lower butyrate, propionate and acetate concentration in IBS‐C mice when compared to controls. To determine contractile properties of intestine after introduction of control and IBS‐C microbiota, we stimulated colonic rings with carbachol and KCl, and observed hypercontractility in IBS‐C microbiota recipients. To determine mechanism for delayed GI transit we analyzed colon tissues in fecal microbiota recipients by qRT‐PCR, and determined dysregulated neuromediator pathways. Overall, these results suggest that IBS‐C dysbiosis contributes to development of delayed transit time through microbial metabolite production and changes in colonic contractile properties.

Support or Funding Information

R01 DK097268 and T32 DK007074
The Baylor KOMP2 Program and BaSH Consortium: A Resource for Producing and Phenotyping Knockout Mice
Authors: Reynolds Corey L , Beaudet Arthur , Dickinson Mary , Seavitt John , Heaney Jason
Published: November 2023

The Knockout Mouse Phenotyping Program (KOMP2) was launched in the Fall of 2011 with the goal of having three mouse production and cryopreservation centers generate a knockout mouse strain for every protein coding gene by using embryonic stem cell resources generated by the International Knockout Mouse Consortium (IKMC). In addition to the generation of these knockout lines of mice, each center is responsible for the systematic, broad‐based, phenotyping of each line using standardized procedures. Phenotyping procedures start when the animal is nine weeks of age and conclude at sixteen weeks of age. The tests include behavioral, metabolic, cardiovascular, auditory, ocular, and histopathological assays. Gene‐to‐phenotype associations are made by a versioned statistical analysis with all data (quantitative and images) freely available on a web portal which offers several data download features. New technology development has been supplemented throughout the projects lifespan to include the standardized phenotyping of embryos from embryonic lethal mouse lines, usage of CRISPR/Cas9 technology to increase productivity and a subset of lines will be examined for small molecule metabolites to detect metabolic abnormalities. At present, the KOMP2 project has produced over 2,500 lines of mice and phenotyping of these lines is on track to be completed by Fall 2016 with all data currently being freely available to the scientific community.

Support or Funding Information

NIH U54‐HG006348