bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficacy of Tolerizing Vaccination Strategy for Mitigating Autoimmune‐Mediated Cardiac Injury
Authors: Lust Robert M. , Reece Shaun P. , Van Scott Michael R. , Mannie Mark D. 
Published: November 2023

BackgroundInfectious agents that induce cross‐reactive myocarditic T cell responses may cause autoimmune inflammation of the heart, directly causing cardiac dysfunction, or they may produce an undetected autoimmune background that could manifest later as an exacerbating influence in the setting of future myocardial injury.PurposeThis study was designed to evaluate the efficacy of GMCSF‐myosin fusion proteins as tolerogenic vaccines capable of inhibiting experimental autoimmune myocarditis (EAM).MethodsThe fusion proteins were comprised of an N‐terminal granulocyte macrophage‐colony stimulating factor (GM‐CSF) domain and a C‐terminal cardiac myosin antigen domain. The antigen domains Myosin 1052–1076 and myosin 614–643 were chosen based on epitopes known to cause EAM in Lewis rats and Balb/c mice respectively. Bioactivity of the cytokine domains for GMCSF‐Myo1052 and GMCSF‐Myo614 was confirmed with bone marrow proliferation assays. Enhanced antigen presentation was confirmed for GMCSF‐Myo1052.ResultsBoth fusion proteins were found to inhibit the development of EAM when given as a pretreatment before EAM induction. Myo1052 fusion protein reduced the inducible incidence of EAM by nearly half, from 81.3% (13/16) to 46.7% (7/15), and the severity of EAM by 62%, from a histology severity score of 1.56 ± 1.41 to 0.60 ± 0.83 (mean ± SD, 5 point scale). Myo614 fusion protein reduced the inducible incidence of EAM by 67%, from 76.9% (13/16) to 25% (3/12), and the EAM severity by 85%, from a histology severity score of 2.23 ± 1.42 to 0.33 ± 0.65. Additionally GMCF‐Myo614 showed promise for inhibiting EAM when given after EAM induction. Serial assessment of myocardial performance was completed using echocardiography. Interestingly, despite the histological changes, cardiac function was preserved throughout. A transient increase in wall thickness was observed between days 14 and 17, but had resolved by day 21. Coupled with evidence of pericardial effusion, and in the context of the later histology findings, the change in wall thickness was interpreted to suggest building intramural myocardial edema over the second week that had resolved by the end of the third week. To explore the potential use of knockout mouse strain to identify mechanisms associated with fusion protein tolerizing efficacy, EAM was induced in C57BL/6 mice (the background stain for most knockout lines) using a peptide (myosin 718–736) derived from the murine cardiac myosin heavy chain and theorized to bind to IAd. Myosin 718–736 was capable of inducing EAM in Interferon Gamma receptor knockout mice on the C57BL/6 background. Additional studies revealed nitric oxide was not the primary effector molecule responsible for EAM resistance in C57BL/6 wild type mice.SummaryThese studies identify a novel potential means for reducing autoimmune mediated cardiac injury.
Inhibition of Allograft Inflammatory Factor‐1 Attenuates B Cell Development and Infiltration to Cardiac Tissue During the Development of Diabetic Cardiomyopathy
Authors: Sarkar Amrita , Shukla Sanket Kumar , Sikder Kunal , Tsygankov Alexander Y. , Rafiq Khadija 
Published: November 2023

ObjectiveDiabetic cardiomyopathy (DCM) is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. The disease is characterized by intramyocardial inflammation, cardiomyocytes apoptosis, and cardiac fibrosis, and it can lead to heart failure. The molecular mechanism that links inflammation to DCM is incompletely understood. This study investigates the novel role of allograft inflammatory factor‐1 (AIF‐1) gene expression over the humoral responses during the progression of DCM.Methods and ResultsTen‐week‐old wild type male mice were injected with streptozotocin (50 mg/kg for 5 days intraperitoneally) and studied 8 weeks after induction of diabetes mellitus. Mice with elevated blood glucose levels (> 300 mg/dL) were classified as having diabetes mellitus and DCM was confirmed through ECG. Induction of diabetes resulted in an increase in inflammatory cells. This increase correlated with myocyte death and cardiac contractile dysfunction, indicating a possible role of immune cells in the development of heart failure. We therefore assessed which immune cells were involved in myocyte apoptosis. We found that STZ‐induced diabetes leads to an increase of neutrophil infiltration, which is associated with a decrease of B cell growth and function as well as an increased level of AIF‐1. STZ‐induced diabetes also leads to an increase of interleukin 21 (IL‐21), which is known to induce apoptosis in both resting and activated B cells, followed by a decrease of BCl2 and BCl‐xL expression. Our data strongly demonstrated that decreased AIF‐1 expression is associated with downregulation of B cells and that AIF‐1 mediates migration of B cell in vitro.ConclusionsDownregulation of the B cell/AIF‐1 interaction leads to myocyte apoptosis and contributes to adverse cardiac remodeling after diabetes induction. These findings point to AIF‐1 as a potential therapeutic target for the maintenance of cardiac function and remodeling during the development of DCM.Support or Funding InformationThis work was funded by NIH grant for Dr. Khadija Rafiq.
Modulation of Heme Biosynthesis by Ferrochelatase Inhibition Controls Soluble Guanylate Cyclase Expression and Superoxide Production in Bovine Coronary Arteries
Authors: Alruwaili Norah , Sun Dong , Wolin Michael S 
Published: November 2023

Nitric oxide interacts with the Fe2+ heme prosthetic group in soluble guanylate cyclase (sGC) stimulating the conversion of guanosine 5′‐triphosphate to cyclic guanosine 3′,5′‐monophosphate (cGMP). Adequate cGMP production is associated with protective mechanisms against multiple cardiovascular disease processes. Thus, disturbances in this signaling pathway can lead to many pathological consequences. Since there has been minimal investigation on the consequences of altering heme biosynthesis on the cardiovascular system, we investigated if mitochondrial heme biosynthesis is an important factor in controlling the expression and function of sGC and systems influencing superoxide generation and actions. For the purpose of this study, we induced a heme deficiency by inhibiting ferrochelatase (FECH) with N‐methyl protoporphyrin IX (NMPP), potent FECH inhibitor. Bovine coronary arteries (BCA) were treated for 24 hours with 10 μM NMPP. Chemiluminescence (5μM lucigenin) was used to measure superoxide production; western blot analysis was used to measure expression of FECH, sGC and other heme‐containing proteins; and a QuantiChrom heme assay kit was used to measure heme levels. NMPP treatment promoted sGC depletion and attenuation of its activity based on it decreasing protein kinase G‐mediated phosphorylation of VASP and attenuation of relaxation to a nitric oxide donor. NMPP also decreased the expression of heme proteins including cytochrome oxidase subunit 4 and catalase. In addition, superoxide production was significantly elevated in NMPP treated coronary arteries when compared to that in control samples, and scavenging extra mitochondrial superoxide with Tempol and mitochondrial matrix superoxide with MitoTempol attenuated the increase in superoxide. NMPP stimulated increases in superoxide were associated with a decrease expression of mitochondrial FECH expression. Thus, inhibition of FECH increased extra mitochondrial and mitochondrial superoxide and depleted sGC. Our studies suggest that disruption of heme biosynthesis resulting in a loss of cGMP production may serve as a contributing mechanism to the progression of cardiovascular disease.Support or Funding InformationFunded by NIH grants R01HL115124 & R01HL129797
Repetitive ROS Inhibits Nrf2 Antioxidant Defense in Ischemic Heart Failure
Authors: Benipal Simranjeet Singh , Liu Tingting , Knowlton Anne 
Published: November 2023

Nrf2, the master regulator for the antioxidant response, is critical for protecting the heart against reactive oxygen species (ROS). Normally, Nrf2 is bound to Kelch‐like ECH‐associated protein 1 (Keap1) and ubiquitinated in the cytosol. With increased oxidative stress, Nrf2 is released from Keap1, and moves into nucleus, binding to the antioxidant response element (ARE) in target gene promoters. This results in increased expression of antioxidant genes. Ischemic heart failure (IHF), a major cause of cardiovascular morbidity and mortality, is an inflammatory, chronic disease characterized by increased oxidative stress and progressive loss of ventricular function; however, we found Nrf2 expression was decreased by 50% in this disease. The interesting question is does Nrf2 inactivation with corresponding failure of antioxidant response contribute to the progression of IHF? We hypothesized that repetitive ROS exposure in IHF results in depression of Nrf2 activity, which as Nrf2 also regulates its own expression will in turn lead to decreased Nrf2. H2O2 is a major contributor to oxidative stress and damage thus is ideal to investigate the circumstances that occur in vivo due to ROS. Repetitive ROS treatments (modeling the chronic condition) reduced Nrf2 levels by 50% compared to a single treatment. In experiments with isolated rat adult cardiac myocytes treated with repetitive ROS treatments, immunohistochemistry revealed Nrf2 failed to traffic to the nucleus. Downregulation of Nrf2 in IHF and in isolated adult cardiac myocytes after repetitive ROS treatment was associated with decreased Nrf2 nucleus binding activity, suggesting inhibition of the Nrf2 mediated antioxidant defense. Further work evaluated changes in gene expression and mitochondrial function with the reduction of Nrf2.Support or Funding InformationThis work was supported by the following: APS Undergraduate Research Fellowship (UGSRF); NIH RO1 HL079071; VA, Merit Review, 5101BX000839; and NIH R01 HL085844.
Ablation of Toll Like Receptor 4 Mitigates Cardiac Mitochondrial Dysfunction in Hyperhomocysteinemia
Authors: Jeremic Nevena , Metreveli Naira , Tyagi Suresh Chand 
Published: November 2023

BACKGROUNDTLR‐4 is a foreign antigen sensor that plays role in innate immune system activation and has recently gained a significant attention in the field of hypertension but still very little or nothing is known regard regulation of TLR expression within the heart, although TLR4 appears to be upregulated in the failing human heart. Mitochondrial dysfunction and mitochondria‐dependent apoptosis have been shown to promote endothelial cell loss leading to endothelial dysfunction that contributes to pathogenesis of hypertension. These events induce mitochondrial dysfunction characterized by excessive mitochondrial fission and mitochondrial apoptosis contributing to cardiac remodeling followed by hypertension.OBJECTIVEThe objective of this study is to define the mechanisms of homocysteine effect on heart that promote cardiac remodeling and hypertension and explore the role of TLR‐4 mutation in alleviation of homocysteine negative effects.METHODSFor this study we used 5 groups of mice: C57BL/6J, C3H/HEouJ, CBS+/−; C3H/HeJ, and CBS+/−/C3H. For further analysis we used isolated heart and collected blood. We collected physiological data such as: ultrasonography of heart, cardiomyocyte contractility and blood pressure. We checked expression of mitochondrial fusion and fission proteins, antioxidant markers and expression of collagen/elastin fragments.RESULTSData showed that there were increased values of systolic and diastolic pressure in CBS+/− mice (DP: 128.09±17.11; SP: 162.31±12.55) and C3H/HeJ mice had decreased levels of in comparing to other groups (DP: 51.82±14.82; SP: 111.38±6.40). Tau was increased in C3H/HeJ compared to CBS+/−/C3H mice and time to pick was increased in C3H/HeJ compared to CBS+/− mice. Fusion and fission ratio (Mfn2/DRP1) were increased in C3H/HeJ mice (0.98±0.06).CONCLUSIONThese findings indicate the prevalence of mitochondrial fission over mitochondrial fusion in HHcy may explain possible endothelial cell loss and dysfunction followed by collagen accumulation that contributes to cardiac remodeling.Support or Funding InformationGrant: NIH (HL74185)
eNOS Regulation of Nrf2 and BNIP3 in Primary Murine Hepatocytes
Authors: Meers Grace M , Naik Radheya Y , Morrison Austin G , Sheldon Ryan D , Rector R. Scott 
Published: November 2023

It is well known that endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) stimulates mitochondrial biogenesis in a cGMP‐PGC1α dependent manner in various tissues. However, the importance of eNOS in regulating mitophagy, the clearance of poorly functioning mitochondria, in hepatocytes remains unknown. We hypothesized that eNOS induces mitochondrial turnover through nuclear factor‐E2‐related factor‐2 (Nrf2) mediated BCL2/E1B 19 kDa protein‐interacting protein 3 (BNIP3) induction of mitophagy. To examine the relationship between eNOS, Nrf2, and BNIP3 in primary hepatocytes, we isolated primary hepatocytes from C57/BL6 wild‐type (WT) mice followed by siRNA treatment [Scramble control (Scr), eNOS, or Nfr2] for six hours. Growth media was applied for 12–18 hours after siRNA treatment, then 24 hours of either control media or 500 μM FFA (250 μM oleate + 250 μM palmitate); the 500 μM FFA condition also included one of the following treatments for 16 hours: 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, to depolarize mitochondria and induce mitophagy)/10 μM cholorquine (CQ, lysosomal inhibitor), 30 nM CDDO‐Methyl Ester (CDDO‐Me, NRF2 activator), or 50 μM diethylenetriamine (DETA) NONOate/CCCP/CQ. Silencing of eNOS in primary hepatocytes (50% reduction vs Scramble control, p<0.01) knocked down Nrf2 mRNA expression by ~40% and Nrf2 activation (NQO1 mRNA; target gene of Nrf2) and BNIP3 mRNA expression by ~40% following FFA challenge (p<0.05 vs Scr control). In contrast, treating primary hepatocytes with the NO donor DETA NONOate induced Nrf2 activation (NQO1 mRNA) and BNIP3 mRNA by 50% and 300%, respectively (p<0.05 vs Scr control). In support of Nrf2 regulating BNIP3, we provide data, that to our knowledge, is the first evidence that the direct knockdown of Nrf2 in primary hepatocytes by an siRNA approach (−80% knockdown of Nrf2 vs Scr control, p<0.001) not only reduces Nrf2 activity (−80% knockdown of NQO1 mRNA), but also blunts induction of mitophagy flux (BNIP3 induction with CCCP/CQ reduced 50% vs Scr) in primary hepatocytes. In addition, Nrf2 activation with CDDO‐Me treatment (3.5‐fold increase in NQO1 mRNA) induced a ~2‐fold increase in BNIP3 accumulation (w/CCCP+CQ), an effect completely ablated with Nrf2 siRNA. These preliminary observations point to a regulatory role of hepatocyte‐specific eNOS in Nrf2 and BNIP3, and a strong rationale for the further examination of the role of Nrf2 in the regulation of BNIP3 and mitophagy.Support or Funding InformationFunding: This work was supported by grants from the Missouri Foundation for Veterans Medical Research, the Richard Wallace Faculty Incentive, Department of Medicine Research Council, and a Veterans Affairs VHA‐CDA2 Grant (IK2BX001299).
Effects of WIN 55,212‐2 and AM281 in mitochondrial respiration of skeletal muscle
Authors: Sanchez‐Duarte Elizabeth , Trujillo Xochitl , Huerta Miguel , Rios‐Silva Monica , Sanchez‐Briones Luis Alberto , Ortiz‐Avila Omar , Saavedra‐Molina Alfredo , Montoya‐Perez Rocio 
Published: November 2023

The endocannabinoid system is emerging as a critical agent of cellular metabolism regulation, through its effects on the cellular respiration and energy production. Most of the cannabinoid and endocannabinoid effects have been mediated predominantly via the activation of the cannabinoid type 1 (CB1) receptor, which is widely distributed in the brain and peripheral organs as the skeletal muscle and recently were confirmed to be localized on the outer mitochondrial membrane of these tissues. It is known that their activation by synthetic cannabinoids is able to cause changes in mitochondrial respiration of brain mitochondria, however, little is known about its effect in skeletal muscle. Thus, the aim of this work was to explore the effects of the cannabinoid agonist WIN 55,212‐2 and the CB1 antagonist receptor AM281 on oxygen consumption in the state 3 from mitochondrial respiration in isolated mitochondria derived from rat skeletal muscle.MethodsMitochondria from hindlimb muscles of male Wistar rats weighing between 300 and 350 were isolated by differential centrifugation. The rate of oxygen consumption was determined in a sealed glass chamber with constant stirring at room temperature, using a Clark‐type oxygen electrode coupled to an oxygen monitor YSI 5300 and a computer for data acquisition. The determinations started after adding 10 mM glutamate/malate as respiratory substrate for complex I (state 4) and 1 mM ADP was added to determine oxygen consumption in the phosphorylating state (state 3). Finally, the inhibitor of complex IV (1mM KCN) were added to inhibit mitochondrial respiration. To explore the effects of the experimental drugs on mitochondrial respiration, a concentration‐response curve was performed for each of them: WIN 55,212‐2 (0.1, 1, 5 and 10 μM) and AM281(1, 5, 10 and 20 μM).Our results show that both drugs have similar effects by decrease mitochondrial respiration, this inhibitory effects on oxygen consumption is dose‐dependent by WIN 55,212‐2 being more prominent with 10 μM (40%) and to a lesser extent with 0.1 μM (≈ 20%), while with AM281 decreased 50% with 1 μM. These results suggest that the effect by this drugs on mitochondrial respiration exert a similar effect as has been previously reported in brain mitochondria and that could be acting on other respiratory chain components.Support or Funding InformationWe thank to Universidad de Colima, Universidad Michoacana de San Nicolás de Hidalgo (UMSNH) and Consejo Nacional de de Ciencia y Tecnología (CONACyT) the partial financial of this research. ESD was a receipient of a CONACyT PhD fellow. We acknowledge the partial financing to Coordinación de la Investigación Científica of UMSNH: ASM‐CIC‐2.16; RMP‐CIC 182171.
Sex and Metabolic Fuel Preferences Impact Proliferative Capacity of Human Muscle Progenitor Cells
Authors: Riddle Emily S , Roman Heather A , Thalacker‐Mercer Anna E 
Published: November 2023

IntroductionSkeletal muscle (SkM) stem cells, satellite cells (SC), are essential for muscle maintenance as well as repair and regeneration post injury. Successful SkM regeneration requires a well‐orchestrated series of events from activation of the quiescent SC, to proliferation [muscle progenitor cells (MPC)], differentiation, and formation of multinucleated cells. During the regenerative process, macronutrients provide energy and substrates for metabolic processes and cellular structures. Defects in macronutrient metabolism and metabolic inflexibility underlie metabolic disease and likely impact the regenerative process. The “phenotype” of human macronutrient utilization and sex specific differences throughout the initial stages of the regenerative process are largely unknown.ObjectiveThe objective was to characterize metabolic gene expression and signaling throughout human MPC proliferation and identify sex differences that might impact successful regeneration.MethodsPrimary human SC were obtained from the vastus lateralis SkM of healthy young males (male‐MPC, n=6) and young females (female‐MPC, n=6) and cultured. A high‐throughput imaging cytometer was used to determine the confluency, nuclei count, and % of dead cells every 24 hours throughout proliferation. Saturation density was defined as the # of nuclei at proliferation plateau/cm2. Real‐time qPCR was used to measure mRNA abundance of select genes associated with macronutrient uptake and metabolism, mitochondrial biogenesis, inflammation, and stress at the first and third day out of lag. The first day out of lag was defined as the first increase in % confluency of ≥ 5%.ResultsPercent confluency throughout proliferation and saturation density were lower in female‐MPC when compared to male‐MPC. Female‐MPC also had a higher % of dead cells throughout proliferation. PGC1‐α, CD36, and NFkB mRNA levels were higher and IL6, CAT1, and CAT2 mRNA levels were lower in female‐MPC compared to male‐MPC. PGC1‐α and CD36 mRNA levels increased over time while CAT1 mRNA levels decreased over time in both female‐MPC and male‐MPC. No differences in SLC7A5, GLUT1, or GLUT4 mRNA levels were observed between sexes at either time‐point.ConclusionsFemale MPCs proliferated at a slower rate, reached a lower saturation density, and experienced elevated cell death throughout proliferation when compared to males. These differences in markers of proliferative capacity were accompanied by up‐regulation of metabolic genes, CD36 and PGC1‐α. This suggests that female cells may rely more heavily on fatty acids and mitochondrial function during proliferation, which may impair female MPC proliferative capacity.Support or Funding InformationCornell University
The Impact of Disuse Muscle Atrophy on Perivascular Stem Cell Quantity and Function
Authors: Munroe Michael R , Dyle Michael C , Adams Christopher M , Boppart Marni D 
Published: November 2023

BackgroundSkeletal muscle atrophy can occur as a result of disuse following limb immobilization, extended bed rest, and spinal cord injury. The mechanisms underlying the duration and severity of disuse atrophy are multifaceted, and as such, require continued study to develop effective therapies which can combat this debilitating condition. Perivascular stem cells are located in a variety of tissue types and have substantial regenerative potential, yet the effect of disuse on their quantity and functionality within muscle is unknown.PurposeThe purpose of this study was to determine the extent to which hindlimb immobilization, with or without mechanical reload, affects perivascular stem cell quantity within the skeletal muscle of adult mice.MethodsCohorts of three month‐old male C57BL/6 mice were immobilized by unilaterally stapling the foot in full dorsotibial flexion in order to atrophy the tibialis anterior (TA) muscle. Mice either underwent 14 days of immobilization (IM) or 14 days of immobilization followed by 14 days of reload (IM+RE). At the end of each time point, both stapled (ST) and unstapled (US) TA muscles were dissected and weighed. For one cohort of mice, each muscle was then processed for multiplex flow cytometry to analyze NG2, CD146, PDGFRα, CD45, and CD31 content within the isolated mononuclear suspension. A second cohort of mice had TA muscles dissected and frozen in precooled isopentane for histological analysis.ResultsFollowing IM (n=9), TA muscle weight was significantly reduced compared to both contralateral and cage controls (p=0.003). After IM+RE (n=10), TA muscle weights had failed to return to baseline levels and remained significantly reduced compared to controls (p<0.001). The quantity of perivascular stem cells was lower post‐IM (n=4) with a 31% reduction in NG2+CD45−CD31− (p=0.08), a 32% reduction in CD146+CD45−CD31− (p=0.04), and a 51% reduction in PDGFRα+CD45−CD31− (p=0.01) cell quantity compared to the contralateral control. However, no significant differences were detected for any perivascular stem cell population between US and ST limbs following IM+RE (n=5) (p>0.05).ConclusionThese findings indicate that perivascular stem cell quantity is decreased with disuse atrophy, and normal mechanical reload restores quantity to baseline values. Given the responsiveness of these cells to disuse and mechanical stimuli, perivascular stem cells are viable therapeutic targets in the treatment of skeletal muscle disuse atrophy.