bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glucocorticoid Receptor Signaling is Critical for microRNA Gender‐Specific Regulation of Gene Expression in the Adult Mouse Heart.
Authors: Cruz‐Topete Diana , Oakley Robert H , Xu Xiaojiang , Cidlowski John A 
Published: November 2023

Heart disease remains a leading cause of death among women in the US. Stress is considered a risk factor for heart disease. Growing evidence indicates that women are more susceptible and present higher stress levels at baseline than men, suggesting an important role for stress signaling in female cardiac health. Glucocorticoids are the primary regulators of the stress response and exert their actions by binding the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of transcription factors. GR has been shown to play a direct role in heart physiology, but its sexual dimorphic effects in cardiac gene expression are unclear. microRNAs regulate a plethora of cellular and physiological processes and dysregulation in their expression has been linked to heart disease. However, there are limited studies on gender‐specific differences in microRNAs expression and regulation in the adult heart. Our goal was to determine the contribution of GR signaling to the sexually dimorphic expression of microRNAs and their target genes in the mouse adult heart. To accomplish this objective, we performed a RNA‐seq and microRNA array on male and female hearts of the cardiomyocyte‐specific GR knockout (cardio GRKO) and their littermate controls (GR flox/flox). RNA‐seq data revealed that over 6000 genes are differentially expressed between male and female controls hearts at baseline. Data comparison between cardio GRKO and GR flox/flox showed that 3900 (65%) of the identified genes required GR for sexually dimorphic expression. In agreement with the RNA‐seq data, we found that 163 microRNAs were also influenced by sex and that GR was necessary for the gender‐specific regulation of 130 (~80%) of the identified microRNAs. Integrated analysis of microRNA and mRNA expression data revealed that 33 of the 163 identified sex‐dependent microRNAs target the expression of 4200 (70%) of the 6000 genes found differentially regulated in male and female control hearts. The expression of 25 of these 33 microRNAs depends on GR. Among the GR‐dependent microRNAs, we found miR 1–3p, miR‐29, miR‐128 and miR‐370, which are important biomarkers of heart failure in humans. Paired analysis of these 4 microRNAs and the RNA‐seq data, showed that 600 genes associated with cardiac hypertrophy, heart failure and hypoxia are targeted by these microRNAs. Among these genes, we found collagen type I alpha 1 (COL1A), epidermal growth factor receptor (EGFR), angiopoietin 2 (ANGPT2), myocardin (MYOCD) and endothelin receptor type B (EDNRB). These results suggest that GR regulation of microRNAs is a potential mechanism by which stress selectively modulates gene expression and consequently influences cardiac health in women.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Institutes of Health, NIEHS.
The Uremic Metabolite, Trimethylamine‐N‐Oxide (TMAO), Directly Alters Cardiac Contractile Function
Authors: Oakley Carlee , Hendrix Matt , Grillo Michael , McCarthy Darla , Stubbs Jason R. , Wacker Michael J. 
Published: November 2023

BackgroundTMAO is produced by intestinal microbial metabolism of phosphatidylcholine. Clearance of this uremic metabolite is dependent on urinary excretion. Therefore, as kidney function declines during chronic kidney disease (CKD), plasma levels of TMAO increase. Recently, elevated TMAO plasma levels have been reported to show concentration‐dependent associations with cardiovascular disease. However, there have been no experiments to determine the direct effect of TMAO on cardiac muscle. Therefore, our objective in this study was to determine if TMAO directly alters cardiac contractility and intracellular calcium levels.MethodsCD1 adult mouse hearts were extracted, attached to a force transducer, oxygenated, and paced within an organ bath. Changes in contractility were measured after diffusing TMAO through the organ bath. As a follow‐up approach, mouse hearts were reverse perfused through the aorta via a modified Langendorff apparatus to facilitate TMAO tissue perfusion. To further explore a mechanism of action, embryonic cardiac myocytes were isolated and cultured to conduct epifluorescence calcium imaging experiments.ResultsAcute treatment with TMAO, in the diffusion‐based model, increased contraction amplitude of ventricular muscle 17% and 46% at 300 μM (P=0.08) and 3000 μM (P<0.05), respectively. TMAO also increased the slope and area under the curve of the contractile waveform compared to vehicle (n=4–6). Langendorff perfusion of hearts with 300 μM TMAO generated a greater response and increased average contraction height 34% (P<0.05; n=3). TMAO, at 300 and 3000 μM, also increased intracellular calcium levels in spontaneously beating cardiomyocytes.ConclusionsTMAO directly increases cardiac contractility and intracelluar calcium levels. Acutely, these actions may be an adaptive response. Chronically, however, increases in intracellular calcium and cardiac stress promote cardiac remodeling and heart disease. Therefore, TMAO may be a novel contributor in the development of heart disease in CKD and thus an important therapeutic target for reducing morbidity and mortality.
Cardiac Hypertrophy and Myocyte Remodeling Are Exacerbated with Multiple Pregnancies
Authors: Wagner Marcus , Nelson Aaron , Flake Caela , Eaton Deborah , Bailey Beth A 
Published: November 2023

BackgroundThe female heart is adapted to support the hemodynamic load of pregnancy; it hypertrophies during pregnancy then returns to its pre‐pregnancy size relatively soon after delivery. This cardiac hypertrophy has generally been categorized as physiologic hypertrophy similar to that seen with exercise, however a reduction in cardiac function in late pregnancy has been suggested. In addition, the hemodynamic stress of pregnancy can induce a maladaptive, pathologic hypertrophy in a small number of women, and multiparity is thought to increase the risk for this maladaptive response. Because prolonged, persistent hemodynamic stress is associated with the development of pathological hypertrophy, multiple pregnancies may exacerbate any contractile changes seen in a single pregnancy. This study seeks to characterize the contractile properties of the pregnant myocardium during the first versus the third pregnancy.Methods and ResultsLate Pregnancy (LP) Female Swiss‐Webster mice were bred then studied at near term (Embryonic day 17–19). Multi‐pregnant mice (MP) were bred two times and allowed to wean each litter for 3 weeks then bred a third time and studied at embryonic day 17–19. LP and MP were compared to age‐matched, non‐pregnant (NP) controls. Significant cardiac hypertrophy occurred in LP vs NP (253 ± 7mg vs 216 ± 9mg, p<.05), and MP hearts were significantly larger than both LP and NP (339 ±18 mg, p<.01). Individual cardiac myocytes were isolated using collagenase‐based perfusion technique. Sarcomere length (light diffraction) and Ca2+ transients (fluo‐3) were measured at pacing rates of 1 Hz and at bath [Ca] of 1.5mM. Duration of twitch contraction was prolonged (p<.05) in MPM as measured by Time to 75% Recovery (.62 ± .05 vs .43 ± .04 sec in NPM). While twitch duration was prolonged in LPM (.53 ± .04 sec), the difference did not reach significance. Interestingly, the duration of the fluo‐3 calcium transients were similar in LPM (.24 ± .01 sec) and NPM (.25 ± .02 sec), but the MPM calcium transient duration (.34 ± .03 sec) was significantly prolonged (p<.05) compared to both NPM and LPM. There were no differences in other contractile parameters measured or in the fluo‐3 calcium transient properties. 10−7M Isoproterenol (ISO) was used to determine the responsiveness to adrenergic stimulation. ISO induced significantly enhanced contractility in NPM, LPM, and MPM, and the response was heightened in LPM and MPM such that the presence of ISO normalized the differences in the durations of twitch contractions between NPM, LPM, and MPM.ConclusionsThese results suggest that the cardiac hypertrophic response to pregnancy and subsequent alterations in contractile duration are exacerbated after multiple pregnancies. Enhanced response to ISO normalizes the contractile differences and suggests and increased cardiac reserve in LPM and MPM.Support or Funding InformationHoward Hughes Medical Institute American Physiological Society
Endurance Exercise is Associated with Decreased Calpain to Calpastatin Ratios in Aged Rat Hearts
Authors: Sepe Joseph J , Hacker Timothy A , Diffee Gary M 
Published: November 2023

The Ca2+‐activated protease calpain has been shown to play a deleterious role in the heart during the aging process, which ultimately impacts cardiac function. Calpastatin is an endogenous inhibitor of calpain, and the ratio of calpain to calpastatin in cardiac myocytes is physiologically important because this ratio greatly impacts the ability of Ca2+ to activate calpain. It has been suggested that activation of calpain may lead to proteolytic degradation of sarcomeric proteins, which ultimately contributes to impaired contractile function in the aging heart. Exercise training has been shown to mitigate and even reverse many aspects of the aging process in the heart. However, little is known about the effects of endurance exercise training on cardiac levels of calpain I, calpain II, or calpastatin in senescent hearts. To address this question, we conducted a 3‐month endurance treadmill training study in aged rats. Exercise capacity (as measured by time to exhaustion, EX‐TIME) and VO2max were measured in 22 month old rats and then animals were divided into Sedentary (SED) vs. Exercise (EX) groups. EX animals were trained 5 days a week for 3 months, with speed and duration gradually increased to the point that animals were running at 14 m/min for 45 min. At the end of the 3‐month training period, exercise capacity and VO2max were again measured in all animals. Mean EX‐TIME and VO2max values significantly (p <0.05) declined in SED animals from Pre‐training (PRE) to Post‐training (POST). EX‐TIME declined by 58% in POST compared to PRE, while VO2max declined by 21% in POST vs. PRE. In the EX animals there were significant (p <0.05) increases in EX‐TIME (POST values 172% of PRE) and VO2max (POST values 141% of PRE). To assess cardiac function, echocardiography was used to measure Ejection Fraction (EF%) and Fractional Shortening (FS%). EX training significantly (p <0.05) improved EF% and FS% compared to SED animals (EF% was 36% greater in EX compared to SED; FS% was 26% higher in EX compared to SED). Following sacrifice, rat hearts were quickly excised, and the left ventricle was dissected and flash frozen for Western blot analysis. Endurance exercise training was associated with decreased calpain to calpastatin ratios in aged hearts compared to sedentary counterparts (p <0.05). There was no significant difference in calpain II levels between EX and SED hearts. The improved calpain/calpastatin ratio exhibited by EX animals appears to be due to a significant decrease in calpain I levels compared to SED animals (p <0.05). These results show that endurance exercise increased cardiac function in senescent rats, and that this increase was associated with a decrease in the ratio of calpain to calpastatin. Changes in the cardiac levels of calpain I appear to underlie the decrease in this ratio.Support or Funding InformationMarsh Center for Research in Exercise and Movement, University of Wisconsin‐Madison
5‐Methoxytryptophan Promotes Endothelial Cell Regeneration After Vascular Injury
Authors: Ho Yen‐Chun , Chen Chung‐Huang , Wu Meng‐Ling , Ho Hua‐Hui , Liang Li‐Yu , Yet Shaw‐Fang 
Published: November 2023

5‐Methoxytryptophan (5‐MTP) is a metabolite of tryptophan metabolism. We recently demonstrated that 5‐MTP protects against intimal hyperplasia in a mouse neointima formation model of left common carotid artery ligation. The novel protective function of 5‐MTP is in part through reducing vascular smooth muscle cell proliferation and migration. In this study, we investigated the effect of 5‐MTP on endothelial cell (EC) function in response to vascular injury. We subjected approximately 12 weeks old C57BL/6 mice to a femoral artery guide wire injury model to denude endothelium. Following surgery, mice were treated with vehicle (PBS) or 25 mg/kg of 5‐MTP by intraperitoneal injection 3 times a week. Femoral arteries were harvested at early time points after injury for histological analysis to evaluate endothelium. CD31 staining revealed that endothelium was partially regenerated in the injured femoral arteries of vehicle‐treated mice. In comparison, 5‐MTP significantly increased endothelial regeneration in the injured arteries. Given that proinflammatory cytokine IL‐1β and TNF‐α are increased in the injured vessel wall, we next examined whether 5‐MTP could protect against EC dysfunction induced by IL‐1β or TNF‐α. Interestingly, 5‐MTP did not affect IL‐1β or TNF‐α‐reduced EC proliferation. In contrast, 5‐MTP ameliorates IL‐1β‐induced inflammatory cell adhesion and TNF‐α‐reduced EC migration. Taken together, our results suggest an important role of 5‐MTP on EC regeneration after vascular injury.Support or Funding InformationThis work was supported by the Ministry of Science and Technology of Taiwan grants MOST 104‐2321‐B‐400‐002 and 104‐2320‐B‐400‐006‐MY3, and National Health Research Institutes of Taiwan grant CS‐106‐PP‐05
Microparticle Uptake by Pulmonary Microvascular Endothelial Cells
Authors: Scruggs April K , Rich Thomas C , Bauer Natalie N 
Published: November 2023

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Furthermore the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry proteins and RNAs from parent cells and are extracellular packets of information that affect target cell function. However, the mechanisms of targeting and delivery of the message are unknown. Understanding the message delivery system between MPs and target cells is the focus of this project.We first tested whether MPs are internalized by rat pulmonary microvascular endothelial cells (PMVECs) in culture. Cells labeled with Hoechst nuclear stain were incubated with calcein green and PKH26 labeled MPs for four hours. The cells were imaged using an environmental chamber on a confocal microscope (Nikon A1R). An image stack (Z‐stack) was acquired every 15 minutes. MPs were observed within cells and the number of MPs within cells increased over the first 90 minutes of the experiment. Maximum MP‐cell association was observed at 1 hour and few MPs were observed inside cells after 3 hours. These results indicate that MPs are taken up by target PMVECs in a time‐dependent manner.We next examined MP localization with organelles within cells. Organelle markers included monoclonal antibodies against early endosomes (Rab5 and EEA1), lysosomes (LAMP1), cis‐golgi (GRASP65) and trans‐golgi (TGN65), as well as labels for mitochondria, microtubules and cell nuclei. MPs were isolated from cell culture media via serial centrifugation and stained with a lipophilic membrane stain (PKH26). PMVECs were treated with stained MPs for 1, 2, 4 and 6 hours before organelle labeling. At one hour MPs colocalized with early endosomes (EEA1) and a small percentage colocalized with lysosomes (LAMP1). At six hours MPs colocalized with trans‐golgi. No overlap between MP‐staining and cis‐golgi, mitochondria, microtubules, or nuclei was observed at any time point. Taken together, these data indicate that MPs are internalized by target PMVECs. After internalization by target cells, most MPs are trafficked to the trans‐golgi for possible recycling of MP‐contents. Future studies will investigate the mechanisms responsible for internalization, whether payloads of MPs are delivered to specific organelles, and whether MP‐delivered proteins alter target cell function.Support or Funding InformationThis work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.
Endothelium‐Independent Vasorelaxation Induced by Nitric Oxide Generation from the Novel Organic Nitrate NDOP Is Partially Mediated by Inward‐Rectifier Potassium Channels
Authors: de Paula Ricardo B , Montenegro Marcelo F , França‐Silva Maria S , Brandão Maria C , Diniz Thiago F , Lemos Virginia S , Athayde‐Filho Petrônio F , Magnani Marciane , Lundberg Jon O , Carlström Mattias , Braga Valdir A 
Published: November 2023

Nitric oxide (NO) deficiency is crucially linked to the development and progression of cardiovascular diseases, including arterial hypertension. Here, the mechanisms underlying the vascular effects of the new organic nitrate 2‐nitrate‐1,3‐di(octanoxy)propane (NDOP), a putative nitric oxide (NO) donor, were investigated. A combination of in vitro and ex vivo approaches was used to assess the effects of NDOP on vascular reactivity and NO release in normotensive C57BL/6J mice. The intracellular fluorescent probe DAF‐2 DA revealed NDOP‐mediated NO formation in isolated vascular smooth muscle cells from mice aortae (53.2 ± 1.6 vs. 10.7 ± 0.9 a.u., n = 13, P < 0.05). The increased NO signal by NDOP was not affected by inhibition of endothelial nitric oxide synthase (eNOS). In isolated phenylephrine‐preconstricted aortic ring preparations, NDOP‐mediated vasorelaxation (10−8–10−3 M) was not reduced by previous inhibition of eNOS (112.5 ± 7.4 vs. 107.3 ± 7.5%, n = 7). However, inhibition of soluble guanylyl cyclase with ODQ (22.2 ± 6.7, n = 6, P < 0.05) and treatment with the NO scavengers PTIO (75.7 ± 5.6, n=7, P < 0.05) and PTIO+hydroxocobalamin (38.8 ± 4.6, n = 4, P < 0.05) all reduced NDOP‐mediated endothelium‐independent vasorelaxation in aortae compared with control. When aortic rings were contracted with modified Krebs solution by KCl (60 mM), NDOP‐induced vasorelaxation was attenuated (80.4 ± 5.2 n = 6, P < 0.05). Non‐selective inhibition of potassium (K+) channels using Krebs depolarizing solution (KCl 20 mM) reduced relaxation induced by NDOP (72.8 ± 3.4; n = 6, P < 0.05) suggesting the involvement of K+ channels. Selective blockade of large conductance calcium‐activated potassium channels (BKca) by tetraethylammonium (1 mM), voltage‐gated potassium channel (Kv) by 4‐aminopyridine (1 mM) or ATP‐sensitive potassium channels (KATP) by glibenclamide (10 μM) did not affect relaxation induced by NDOP. However, pre‐incubation with barium chloride (30 μM), an inward‐rectifier potassium channels (KIR) blocker reduced NDOP‐mediated vasorelaxation (73.7 ± 5.7, n = 6, P < 0.05) compared with control. Direct measurements of NO gas by chemiluminescence demonstrated dose‐dependent NO generation by NDOP (0.1 mM = 1.8 ± 0.2 ppb; 1 mM = 9.0 ± 0.8 ppb; 10 mM = 17.2 ± 2.3 ppb, n = 3), which was dependent on xanthine oxidoreductase activity. In conclusion, NDOP‐mediated vasorelaxation involves increased NO formation, which can stimulate sGC‐cGMP signaling and alter the function of inward‐rectifier potassium channels.Support or Funding InformationCapes, CNPq and STINT
Phosphorylation and redistribution of Vangl2, a planar cell polarity protein, in endothelial cells in response to laminar shear stress
Authors: Curtis‐Whitchurch Logan , Rekapally Harish , Hoying Jay 
Published: November 2023

In response to fluid flow, vascular endothelial cells (ECs) reorganize intracellular compartments to orchestrated regional signaling responses related to shear‐dependent EC function. While it is known that the EC cytoskeleton is involved in shear‐dependent EC reorganization, less is known about the complex dynamics integrating cytoskeleton reorganization and signaling. Using cell culture and ex vivo systems, we have evidence that human ECs express Vangl2, a highly conserved, transmembrane protein that organizes signaling in epithelial cells via a planar cell polarity mechanism in a phosphorylation‐dependent manner. Given its function as a cell signaling organizer, we hypothesized that Vangl2 plays a similar role in EC, but responds to unidirectional laminar fluid flow, a unique endothelial cell stimulus. Laminar flow (~15 dynes/cm2 for 2 to 48 hours) induced phosphorylation of Vangl2 and segregation into the detergent insoluble fraction in cultured human ECs as compared to static conditions. phospho‐Vangl2 co‐immunoprecipitated with vimentin, a cytoskeleton element associated with shear‐dependent EC polarization, and displayed colocalization in a time‐dependent manner following exposure to laminar flow. Knockdown of either vimentin or Vangl2 via siRNA revealed an interdependent dynamic between the two proteins. Finally, Vangl2 was asymmetrically located in the ECs of the intact mouse aorta, but uniformly distributed throughout the cells in areas of disturbed flow, similar to what we observed in cultured ECs. We report that the highly conserved, cell‐organizing protein, Vangl2 is expressed in ECs and that it differentially regulated by laminar flow. Our findings suggest a role for Vangl2 in organizing signaling systems within ECs in response to shear stress.Support or Funding InformationSupported by the AHA and KSCIRC Neuroscience core facilities that are supported by the NIH/NIGMS P30 GM102507 grant.
Use of Phase Loop Plots to Assess Ca2+ Sensitivity of Force Generation in Airway Smooth Muscle
Authors: Han Young Soo , Sieck Gary 
Published: November 2023

In excitation‐contraction coupling, a transient elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) triggers a force response. The Ca2+ sensitivity of muscle reflects the relationship between [Ca2+]cyt and force, and in skeletal and cardiac muscle is often defined as the [Ca2+]cyt at which 50% force is generated. In smooth muscle, Ca2+ sensitivity has been assessed as the ratio of peak force to peak cytosolic [Ca2+]cyt. However, this measurement overlooks the dynamic nature of Ca2+ regulation of force generation. In the present study, in order to provide a dynamic index of Ca2+ sensitivity in airway smooth muscle (ASM), we simultaneously measured [Ca2+]cyt and force and determined their temporal dependence using a phase loop plot. We hypothesized that [Ca2+]cyt/force phase loop plot in ASM would be altered by conditions in which the extent of regulatory myosin light chain (rMLC) phosphorylation was affected.Intact ASM strips were isolated from porcine tracheas obtained from local abattoir. ASM strips, were loaded with Fura‐2 AM (Ca2+ fluorescent indicator) and stimulated using 1 μM ACh. Isometric force and [Ca2+]cyt responses were simultaneously measured using a Guth Muscle Research System. In the first set of experiments we examined the effect of Y27623, a Rho‐kinase inhibitor, and calyculin A, a protein phosphatase inhibitor that affected Ca2+‐induced rMLC phosphorylation. In a second set of experiments, we examined the effect of cytochalasin D (Cyto‐D), an inhibitor of actin polymerization, which reduces force independent of Ca2+‐induced rMLC phosphorylation.As expected, Y27623 decreased ACh‐induced isometric force but had no effect on evoked [Ca2+]cyt transients. Cyto‐D also decreased force with no effects on evoked [Ca2+]cyt. By comparison, calyculin A increased force with no effect on [Ca2+]cyt. Although these results suggest changes in Ca2+ sensitivity, the phase loop plots showed no significant shift indicative of changes in dynamic Ca2+ sensitivity. These results do not support our hypothesis that dynamic Ca2+ sensitivity in ASM is modulated by the extent of rMLC phosphorylation. Under these experimental conditions, it appears that isometric force generation in ASM is not entirely dependent on [Ca2+]cyt.Support or Funding InformationSupported by NIH grant HL126451 (GCS)