bluuebunny/crossref_metadata_2025 huggingface dataset:
Placental pathology, corticotropin‐releasing hormone (CRH), and fetal growth in the Pregnancy Outcomes and Community Health (POUCH) cohort
Authors: Perng Wei , Talge Nicole M. , Senagore Patricia K. , Holzman Claudia
Published: November 2023

Objective

To examine the associations of five histopathology‐based placental pathology constructs (maternal vascular – obstructive [MVO]; fetal vascular – obstructive [FVO]; maternal vascular – disturbance of integrity [MVI]; fetal vascular – disturbance of integrity [FVI]; maternal vascular – developmental [MVD]) and second trimester corticotropin‐releasing hormone (CRH) levels with birthweight‐for‐gestational age z‐score (BW/GA).

Methods

We studied 1,154 women in the Pregnancy Outcomes and Community Health (POUCH) study, a community‐based pregnancy cohort in Michigan. Using survey weights to account for the POUCH sampling scheme, we conducted multivariable linear regression to quantify differences and 95% confidence intervals (CI) in BW/GA with respect to evidence of placental pathology (yes vs. no), while also accounting for pre‐pregnancy BMI, race, gestational diabetes, and smoking habits.

Results

Median age at enrollment was 26.7 ± 0.2 years. The majority (67.3%) of women were White, and 22% were African American. Mean ± SD BW/GA was 0.13 ± 0.04. In the overall sample, only MVO was associated with BW/GA (−0.53 [95% CI: −0.76, −0.31]). Stratification by preterm delivery status (n = 286 vs. 868) revealed that MVO corresponded with lower BW/GA in both term (−0.52 [95% CI: −0.77, −0.28] z‐scores lower BW/GA) and preterm births (−0.59 [95% CI: −0.99, −0.19] z‐score lower BW/GA), while FVO was related to lower BW/GA among preterm births only (−0.42 [95% CI: −0.72, −0.13] z‐scores). We observed evidence for effect modification by CRH for MVO, FVO, and FVI among preterm births, thus we further explored relations of CRH (Q1 as lowest, Q2+3 as referent, Q4 as highest) with BW/GA by placental pathology status, only among preterm deliveries. Among mother‐infant dyads with MVO and FVO, higher CRH was associated with 0.80 (95% CI: 0.07, 1.53) and 1.05 (95% CI: 0.51, 1.59) z‐scores lower BW/GA as compared to their counterparts with CRH in the middle quartiles. Among participants with FVI, lower CRH was associated with 0.53 (95% CI: 0.12, 0.95) z‐scores higher BW/GA.

Conclusions

Our findings suggest that (1) maternal vascular obstructive complications are related to poor intrauterine growth regardless of preterm birth status, and (2) high CRH may be a biomarker of poor intrauterine growth in pregnancies that culminate in preterm delivery and exhibit placental pathology related to obstruction of the maternal and fetal circulations.
Association between Hormonal Contraceptive Use and Prevalence of Premenstrual Symptoms
Authors: Jarosz Alicia Caroline , Jamnik Joseph , El‐Sohemy Ahmed
Published: November 2023

Background

Hormonal contraceptive (HC) use may be associated with a reduction in some premenstrual symptoms, however, the evidence remains equivocal.

Objective

To determine the prevalence of premenstrual symptoms in a multiethnic population of women and to investigate the association between hormonal contraceptive use and premenstrual symptoms.

Methods

1048 women aged 20–29 years participating in the Toronto Nutrigenomics and Health Study provided data on their premenstrual symptoms and HC use. Severity of symptoms was classified as none, mild, moderate, or severe. Frequencies of premenstrual symptoms were determined as a total and by ethnicity. Logistic regressions were used to calculate the odds ratio (OR) and 95% confidence interval (CI) to analyze the associations between symptoms and use of HCs, adjusting for ethnicity.

Results

The prevalence of individual symptoms differed as follows: cramps (75%), bloating (74%), mood swings (72%), increased appetite (64%), acne (61%), fatigue (55%), sexual desire (50%), anxiety (35%), desire to be alone (33%), depression (28%), headache (26%), confusion (21%), clumsiness (15%), nausea (15%), and insomnia (11%). Prevalence of cramps, bloating, acne, sexual desire, headache, confusion, and nausea differed between ethnicities (P<0.05). For example, cramps were reported by 79% of Caucasians and 84% of South Asians, but only 67% of East Asians. The OR (95% CIs) for experiencing the following moderate/severe symptoms in women using HCs compared with those not using HCs were: cramps 0.48 (0.33–0.70), fatigue 0.64 (0.44–0.95), depression 0.35 (0.19–0.65), desire to be alone 0.32 (0.18–0.58), clumsiness 0.22 (0.06–0.74), confusion 0.21 (0.08–0.54), and anxiety 0.41 (0.24–0.70). Premenstrual symptoms of acne, mood swings, bloating, increased appetite, headache, insomnia, nausea, and sexual desire were not associated with HC use.

Conclusion

This study demonstrates that the prevalence of premenstrual symptoms varies widely between symptoms and ethnicities. Our findings also indicate that HC use is associated with a lower risk of experiencing many, but not all premenstrual symptoms.

Support or Funding Information

Research support: A.C.J. is a recipient of a Natural Sciences and Engineering Research Council Graduate Scholarship
Characterization of Basal and Stimulated Gnrhr Expression in Pituitary Cells
Authors: Janjic Marija M , Bjelobaba Ivana , Tavcar Jovana , Kucka Marek , Tomic Melanija , Stojilkovic Stanko
Published: November 2023

Gonadotropin‐releasing hormone (GnRH) is a potent stimulus for expression of multiple genes in pituitary gonadotrophs, including the gene encoding GnRH receptor (Gnrhr). Here we examined the signaling pathways accounting for Gnrhr expression in vitro, using primary cultures of rat and mice anterior pituitary cells and mouse immortalized LβT2 gonadotrophs. After removal of anterior pituitary glands and cell dispersion, there was decay in Gnrhr expression, which progressed in cultured cells, with the rates not affected by substitution of horse or fetal calf serum with BSA and by application of a GnRHR antagonist. The decay in gene expression reached a steady state level during 40–50 h incubation to about 20% of that observed in vivo and the residual expression was termed basal. Pulsatile as well as continuous application of GnRH restored, at least partially, Gnrhr expression in a time‐dependent manner, with a peak in expression after about 6 h of application. In pituitaries from neonatal and infantile animals, the profiles of GnRH‐induced responses were similar in females and males and the amplitudes of responses were low. During juvenile to postpubertal period, the profiles of GnRH‐induced gene expression were also comparable in cells from both sexes, but the amplitudes of responses were several‐fold higher in female cells. GnRH‐stimulated Gnrhr expression was independent of cAMP‐PKA signaling pathway and was mediated by calcium and protein kinase C signaling pathways; downstream elements of protein kinase C pathways included ERK1/2 but not JNK, p38, or big MAPK. In naïve LβT2 cells (never previously stimulated with GnRH), there was also a substantial basal Gnrhr expression not affected by removal of fetal calf serum for 24 h and by application of a GnRHR antagonist; these cells also expressed functional GnRHR, as indicated by GnRH‐induced calcium signaling. However, application of GnRH was unable to elevate gene expression in LβT2 cells. Pharmacological activation of cAMP‐protein kinase A, calcium, and protein kinase C signaling pathways were also ineffective. On the other hand, basal Gnrhr expression was reduced by inhibition or depletion of classic protein kinase C isozymes in both normal and immortalized pituitary cells. These results indicate that Gnrhr expression is operative in the absence of any hormonal stimulation, probably reflecting intrinsic protein kinase C activity. Physiologically, such activity secures the presence of sufficient number of functional GnRHR needed to up‐regulate Gnrhr expression and initiate the expression of signature and other genes once GnRH is available.

Support or Funding Information

This work was supported by the Intramural Research Program of the National Institute of Child Health and Human Development (ZIA DH000195‐22).
Replacing pork and chicken with herring (Clupea harengus) increases plasma glucosamine and decreases carbon flux through the citric acid cycle in overweight men and women
Authors: Ross Alastair , Vincent Andrew , Savolainen Otto , Lindqvist Helen , Lind Mads V , Sandberg Ann‐Sofie
Published: November 2023

Many national nutrition recommendations recommend reducing meat intake and increasing fish intake, due to the association between higher fish intake and reduced risk of cardiovascular disease. Although many studies have looked at the health benefits of the n‐3 fatty acids abundant in fatty fish, few intervention studies have investigated the short‐term effects of replacing meat with fish. As part of a wider series of studies revisiting the comparative effects of meat and herring on human metabolism, we used GC‐MS/MS metabolomics to investigate the effects of replacing lean chicken and pork with herring in overweight and obese adults. In a researcher blinded randomised crossover study, eleven subjects ate five frozen meals/wk over 4 wk, with the frozen meals containing 150 g each of either pork or chicken fillets, or herring fillets. The diets were similar in macronutrient content except for a higher amount of fat in the herring meals. Major metabolic changes observed in plasma were that glucosamine, glutamine and ornithine concentrations increased after the herring diet (all p<0.05 after four wk), while chicken and pork led to increases of agmatine, citric acid, isocitric acid and oxalic acid (all p<0.05 after four wk). Glucosamine is commonly associated with better joint health, though we are unaware of any association between increasing plasma concentrations of glucosamine and lower incidence of joint‐related diseases or complaints. Chicken and pork increased concentrations of citric and isocitric acid, while herring increased glutamine, suggesting that these two different diets had an effect on central carbon metabolism, with a differential regulatory effect at α‐ketoglutaric acid. A further effect was evident on arginine metabolism, possibly related to flux through the urea cycle, based on higher concentrations of arginine/ornithine after the herring diet, while chicken and pork increased agmatine, an arginine metabolite via arginine decarboxylase. An additional finding was that methyl histidine, previously suggested to be biomarker of meat intake in urine, was found to be higher after two weeks of the chicken and pork diet (p=0.003), though this was no longer significant after four wk (p=0.05). These findings add to our understanding of the wider effects of both fish and meat on human metabolism beyond effects on lipids and underline the importance of protein choice on health.

Support or Funding Information

This work was supported by a Chalmers Life Sciences Area of Advance Grant to A Ross and by grants from Region Västra Götaland, the Swedish National Board of Fisheries (Dr 223‐2451‐01), The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (Grant No 2001‐1246).
The Role of Tight Junction Membrane Dynamics in the Serine Protease‐Induced Increase in Epithelial Barrier Function
Authors: Ronaghan Natalie Jane , Colarusso Pina , Shen Le , Turner Jerrold R. , MacNaughton Wallace K.
Published: November 2023

One of the main contributors to intestinal epithelial barrier function is the composition of the tight junction (TJ) between epithelial cells. In IBD, inflammatory mediators induce disruption of the TJ and may contribute to pathogenesis. We have found that the apical addition of the serine proteases trypsin and matriptase to colonic epithelial cell lines induces a rapid and sustained increase in barrier function. However, the mechanism is incompletely understood. Tight junction proteins are continuously trafficked in and out of the junction to maintain and modulate barrier properties. We aimed to determine if serine proteases alter tight junction dynamics in the membrane using fluorescence recovery after photobleaching (FRAP). Previous studies using siRNA suggest that occludin is an important TJ protein in the serine protease‐mediated increase in TER. EGFP‐occludin was stably expressed in SCBN intestinal epithelial cells, plated on transwells and mounted on a Live Cell Instruments flow chamber, placed on a closed Olympus FV1000 confocal microscope at 37°C, and the chamber perfused continuously on the apical side with heated Krebs that was equilibrated with 5% CO2. Triplicate baseline FRAP runs were conducted along the junction between two epithelial cells strongly expressing EGFP‐occludin. Cells were then perfused apically with trypsin for 15 minutes, and then follow‐up triplicate FRAP experiments were conducted. The mobile fraction, half time of recovery, and direction of recovery were then determined. FRAP recovery curve analysis shows SCBN cells treated with trypsin exhibit significantly increased EGFP‐occludin in the photobleached area after 90 seconds of recovery compared to untreated cells. The mobile fraction is increased but not significantly different (before treatment 58.08 ± 6.99% and after trypsin treatment 73.35 ± 4.59% (SEM) p=0.1164 n=6). Half time of recovery is also not changed (before trypsin 100.8 ± 8.6s and after trypsin 82.42 ± 10.4s p=0.279). From observation and kymograph analysis, there was also no change in direction of occludin recovery, which was from the lateral membrane, after serine protease treatment. Our results suggest that serine proteases may induce an increased trafficking of occludin into the membrane, or change how occludin binds to other tight junction proteins. Future experiments will investigate phosphorylation and binding of occludin with other TJ proteins and the change in membrane dynamics of other TJ proteins such as ZO‐1, tricellulin, and claudin‐1.

Support or Funding Information

CIHR, NSERC
Zonula occludens‐1 ZU5 domain contributes essential stabilizing interactions at the tight junction
Authors: King Jonathan M , Tan Christine Joy P , Thomason Nicole C , White Addison R , Shen Le , Turner Jerrold R
Published: November 2023

Zonula occludens (ZO‐1) is a cytoplasmic scaffold protein that structurally and functionally tethers cytoskeletal components and membrane proteins at the tight junction. Interestingly, ZO‐1 has a poorly characterized ZU5 domain at the carboxy‐terminus that ZO‐2 and ZO‐3 proteins lack. The goal of this study is to examine ZO‐1‐ZU‐5 function in a structurally defined manner. To determine ZO‐1‐ZU5 domain function, EGFP‐ZO‐1, EGFP‐ZO‐1ΔZU5 and EGFP‐ZO‐1 constructs mutated within the ZU5 domain hydrophobic pocket were engineered and stably transfected into MDCK II cells. FRAP analyses show that EGFP‐ZO‐1ΔZU5 rapidly recovers and the mobile fraction (Mf) is elevated compared to EGFP‐ZO‐1. The Mf increases by 30% in EGFP‐ZO‐1ΔZU5 as a 50% decrease in t1/2 is observed. Disruption of the ZU5‐hydrophobic pocket caused similar changes suggesting the ZU5 domain participates in stabilizing ZO‐1 at the tight junction. Macromolecular flux studies demonstrated that ZO‐1ΔZU5 expression enhances leak pathway permeability compared to EGFP‐ZO‐1. These data indicate that the ZU5 domain stabilizes ZO‐1 at the tight junction and contributes to development of the barrier to paracellular flux of small solutes.
The Coordination of Cough and Swallow Behaviors in Humans
Authors: Brown Edward H. , Aslan Sevda C. , Ovechkin Alexander V. , Pitts Teresa
Published: November 2023

It is not currently known if the coordinated motor control of cough and swallow behaviors described in animal models is preserved in humans. We hypothesized that similar relationships exist, namely the increased muscle activation and decreased swallow duration during combined cough and swallow behaviors. Young healthy individuals were recruited, and during the experiments voluntary cough was elicited by asking subjects to cough “like there was something stuck in their throat” and continue until instructed to stop. Swallow was initiated by infusing 3 ml of water into the subject's mouth and instructing them to “swallow whenever you need to.” Cough and swallow were elicited individually as well as together to mimic an “aspiration” response. Cough excitability was measured by recording the airflow taken from the mouth and surface electromyographic (sEMG) activity from the 6th intercostal, rectus abdominus, abdominal obliques, and submental muscles. Swallow excitability was measured by submental sEMG. All subjects produced multiple cough epochs and swallows to the stimuli. In the combined condition swallows occurred during the inter‐bout interval, with increased intercostal and abdominal drive during cough; and an increased submental drive during swallow. These results confirm the theory of a highly cooperative airway protection system in the human. Furthermore, this model could be used as a novel diagnostic tool for dysphagia/dystussia, eliminating the ceiling effect of a single cough trial. Supported by HL 111215, HL 103750, and P30‐GM103507.

Support or Funding Information

Supported by HL 111215, HL 103750, and P30‐GM103507.
Does maternal omega‐3 supplementation have beneficial effect on control of breathing in rat pups?
Authors: Baldy Cécile , Tenorio‐Lopes Luana , Mercier Océane , Pothier‐Piccinin Olivier , Seaborn Tommy , Marc Isabelle , Kinkead Richard
Published: November 2023

AIM

Preterm infants present respiratory instabilities and apneas owing to immaturity of the neural circuits that control breathing. Immaturity of respiratory reflexes such as the hypoxic ventilator response (HVR) and laryngeal chemoreflex (LCR) contribute to respiratory disorders in newborn. Fatty acids omega‐3 (Ω‐3) benefits brain development in several ways, including a reduction of neural inflammation. Thus, we tested the hypothesis that Ω‐3 supplementation (via maternal diet) improves respiratory control in rat pups.

METHODS

Experiments were performed on male and female pups (10–11 days old). Pups received Ω‐3 supplementation from an enriched maternal diet (13g/kg of Ω‐3) from birth to day 10–11. Mothers of control pups received a standard diet (3g/kg of Ω‐3) Whole body plethysmography was performed on intact, unanesthetized pups to measure breathing and metabolic responses at rest and during hypoxia (12% O2; 20min). To evaluate performance of chemoreflexes without the confounding effect of a metabolic response, we also compared the duration of LCR‐induced apneas in anesthetized pups. Immunolabeling (Iba‐1) of microglial cells was used to evaluate the effect of Ω‐3 supplementation on capacity for neuroinflammation in the brainstem.

RESULTS

By comparison with controls, maternal Ω‐3 supplementation effectively raised Ω‐3 levels in the blood and brainstem. In awake pups, Ω‐3 increases the apnea index and metabolic rate, especially in females. Both males and females increase the HVR compared to their controls and decrease the drop in body temperature during hypoxia. In anestethized pups, Ω‐3 reduced 32% the apnea duration elicited by LCR stimulation. Finally, the number of microglia cells in caudal NTS was lower in Ω‐3‐supplemented pups.

CONCLUSION

Ω‐3 supplementation generally improves reflexives ventilatory and metabolic responses. However, the higher apnea frequency observed at rest suggests that the effects of Ω‐3 are not homogeneous. The reduced capacity for neuroinflammation (as indicated by the reduced number of microglial cells) may contribute to the beneficial effect of Ω‐3.

Support or Funding Information

This research was supported by CIHR (RK and IM).
Mitochondrial Dysfunction in Acute Lung Injury is Characterized By Loss of Mitochondrial Calcium Uniporter (MCU)
Authors: Islam Mohammad N. , Gusarova Galina A , Das Shonit , Bhattacharya Jahar
Published: November 2023

Acute lung injury (ALI) associates with loss of lung mitochondrial function. Oxidant damage by mitochondrial H2O2 may cause mitochondrial dysfunction. Since the mitochondrial calcium uniporter (MCU) determines mitochondrial Ca2+ levels, hence function, we considered the possibility that oxidant‐induced MCU impairment might underlie mitochondrial dysfunction in ALI. To induce ALI, we intra‐nasally instilled lipopolysaccharide (LPS, 1mg/kg) or PBS (control). To determine H2O2, we expressed a mitochondrial matrix‐targeted probe, matrix‐roGFP, in alveolar type 2 (AT2) cells in situ. By alveolar micropuncture we loaded mitochondrial Ca2+ dye, rhod 2, or the lamellar body (LB)‐staining dye LysoTracker Red (LTR). In control lungs, H2O2 fluorescence was 145±7 grey levels in AT2 cells, indicating successful probe expression. In LPS lungs, AT2 mitochondrial H2O2 fluorescence was 75±8 (P<0.05), indicating increased H2O2 generation. Immunoblots in AT2 cell mitochondria show that LPS downregulated MCU. However, LPS‐induced MCU downregulation could be reversed in mice over‐expressing human catalase (hmCAT) in mitochondria of AT2 cells. In addition, hmCAT over‐expressing mice reversed LPS‐induced inhibition of mitochondrial Ca2+ entry and surfactant secretion in response to a 15s lung hyperinflation. These data are the first direct evidence that lung exposure to LPS causes mitochondrial dysfunction marked by oxidant‐induced loss of MCU. Taken together, our findings indicate that alveolar MCU is a major determinant of ALI. Strategies aimed at restoring alveolar MCU expression might lead to novel therapy for ALI.

Support or Funding Information

PBF fellowship for MNI and HL122730 for JB
Claudin‐5 Decreases Alveolar Barrier Function in Alcoholic Lung Syndrome by Displacing Claudin‐18 from Tight Junctions
Authors: Schlingmann Barbara Lisa , White StevenClaude Dorsainvil , Molina Samuel , Lynn K. Sabrina , Capaldo Christopher T. , Koval Michael
Published: November 2023

We found that alcohol abuse increases the susceptibility to develop Acute Respiratory Distress Syndrome (ARDS) by impairing alveolar epithelial cell tight junctions. Understanding how tight junction proteins are regulated on a molecular level may identify potential new therapeutic targets for the treatment and prevention of ARDS. We used a rodent model of chronic alcohol ingestion and cultured primary alveolar epithelial cells (AEC) to investigate the influence of alcohol on tight junction formation and function. We found that AECs from alcohol fed rats had disrupted tight junction morphology and were leakier than control AECs. Alcohol‐induced changes to AEC tight junctions were accompanied by increased expression of a claudin family tight junction protein, claudin‐5. Immunofluorescence analysis showed that impaired barrier function of AECs from alcohol fed rats was accompanied by disruption of tight junction morphology, spikes, containing claudin‐18. Quantification of cells containing claudin‐18 spikes showed that alcohol increases spike formation and this was also associated with increased claudin‐5 expression. Claudin‐5 knockdown experiments in alcohol exposed AECs showed that decreasing claudin‐5 improved barrier function and reduced the number of cells containing claudin‐18 spikes. Conversely, increasing claudin‐5 expression in control AECs mimicked the effects of alcohol by increasing claudin‐18 spikes and paracellular leak. Live cell imaging experiments revealed that claudin‐18 spikes are sites where claudin‐containing vesicles fuse and form reflecting enhanced claudin turnover which was associated with decreased barrier function. To elucidate the molecular mechanisms of alcohol dependent changes in tight junction proteins, we performed Stochastic optical reconstruction microscopy (STORM), a form of super resolution immunofluorescence microscopy. The data revealed that alcohol ingestion lead to an increased colocalization between claudin‐18 and claudin‐5 which in turn was associated with a decreased colocalization between claudin‐18 and the scaffold protein ZO‐1. When YFP‐tagged claudin‐5 was expressed in normal AECs there was a decreased co‐localization between claudin‐18 and ZO‐1 suggesting that the interaction between claudin‐5 and claudin‐18 impairs its ability to interact with ZO‐1, thus destabilizing incorporation of claudin‐18 in tight junctions. Moreover, agents that inhibit claudin‐5 function in alcohol exposed AECs increased barrier function and stabilized tight junction morphology. The data suggest that targeting claudin‐5 may provide a therapeutic strategy to improve barrier function in ARDS.

Support or Funding Information

Support: Emory URC, NIH‐NIAAA, NIH‐NHLBI. DAAD
MORPHINE PROTECTS AGAINST CARDIAC DYSFUNCTION DURING TYPE 2 DIABETES BY IMPROVING MITOCHONDRIAL STRUCTURE AND FUNCTION
Authors: Zemljic‐Harpf Alice Eugenie , Hoe Louise See , Schilling Jan Mirko , Nguyen Alexander , Vainshav Yash J. , Peart Jason N. , Mahata Sushil K. , Patel Hemal H. , Headrick John P.
Published: November 2023

Diabetes promotes ischemic heart disease, cardiomyopathy, intolerance to ischemia‐reperfusion (I‐R) injury, and refractoriness to cardioprotection. Evidence suggests that cardiac abnormalities during diabetes involve inflammation, lipotoxicity, posttranslational‐modifications as well as mitochondrial dysfunction and impaired opioid receptor (OR) signaling. We test the hypothesis that a novel sustained OR stimulus may overcome diabetes induced cardiac dysfunction via mitochondrial dependent protection.

In a murine type 2 diabetes mellitus (T2DM) model we investigated effects of morphine on cardiac function, structure and I‐R tolerance, mitochondrial function and ultrastructure. Streptozotozin (75mg/kg i.p.) was administered once in non‐fasted 12‐week old mice, and normal chow was switched to 60% kcal high‐fat diet (HFD). 12 weeks of HFD induced 25% weight gain, hyperglycemia, hyperinsulinemia, glucose intolerance, cardiac hypertrophy, moderate cardiac depression, exaggerated post‐ischemic myocardial dysfunction and death, and abnormalities in mitochondrial respiration, ultrastructure and Ca2+‐induced swelling. Morphine administration for 5 days: 1) improved glucose homeostasis and body weight; 2) reversed cardiac depression; 3) enhanced I‐R tolerance; 4) restored mitochondrial ultrastructure; and 5) improved mitochondrial function. A treatment‐matched caloric restriction regime modestly improved I‐R tolerance in T2DM mice without improving in vivo cardiac function.

These data reveal that morphine treatment restores contractile function and ischemic tolerance in diabetic hearts via improvements in mitochondrial structure and function, implicating OR activation as a potential novel target in managing T2DM‐induced myocardial abnormalities.

Support or Funding Information

This study was supported by grants from the National Institutes of Health, USA (HL091071, HL107200, HL115933, and HL066941), a VA Merit grant (BX001963 and BX000783), and the National Health and Medical Research Council of Australia (481922).
Evaluation of the vasopressor responses of metformin and one analogue of metformin in insulin resistance rats
Authors: Gutierrez‐Lara Erika J , Sanchez‐Lopez Araceli , Navarrete‐Vazquez Gabriel , Centurion David
Published: November 2023

Cardiovascular diseases and diabetes are the leading causes of mortality in the world; both of them are associated with insulin resistance. Metformin improves insulin sensitivity and it is associated with cardioprotection and vasculoprotection. In vitro studies have demonstrated that metformin improves vasorelaxation and normalization of endothelial function during diabetes. Thus, in this study the vasopressor responses induced by sympathetic stimulation or by i.v. bolus injections of the agonists noradrenaline (endogenous ligand), methoxamine (α1) and UK 14,304 (α2) were determined in rats with insulin resistance or control rats pretreated with: (1) metformin; (2) one analogue of metformin (N‐benzylimidodicarbonimidic diamide; EGL‐6M); or (3) its vehicle. For this purpose, rats were treated with fructose (solution 15%) or its vehicle (water; control) for four months. Next, both groups were divided into three subgroups which were administrated with: (i) metformin (50 mg Kg−1day−1), (ii) EGL‐6M (50 mg Kg−1day−1) or its (iii) vehicle (water; 1 mL Kg−1day−1) during 28 days. Tolerance glucose curve (TGC) and insulin levels were determined after administration of glucose (1 g Kg−1, p.o.) in the above subgroups. Animals treated with fructose showed hyperinsulinaemia during TGC, which were reverted by metformin or EGL‐6M. Then, under anesthesia with isoflurane, animals were pithed and prepared to measure blood pressure and heart rate. In animals with insulin resistance, the vasopressor responses to sympathetic stimulation were decreased, while responses to noradrenaline were increased and those induced by methoxamine and UK 14,304 were not modified. In control animals, metformin failed to modify the vasopressor responses induced by sympathetic stimulation or by noradrenaline, methoxamine and UK‐14,304. In contrast, EGL‐6M significantly increased the vasopressor responses to sympathetic stimulation; the same results were showed in the insulin resistance rats. In animals with insulin resistance, metformin decreased vasopressor response to noradrenaline but did not modify the sympathetic stimulation responses. These results suggest that insulin resistance modified the vasopressor responses by inhibiting sympathetic tone and probably as a compensatory mechanism it may increase α‐adrenoceptor expression. Moreover, during insulin resistance, the analogue, but not metformin, improved sympathetic neurotransmission in the systemic vasculature.

Support or Funding Information

The authors acknowledge to Conacyt for their financial support.
A Novel Treatment for Late‐Stage Metastatic Carcinoma: Targeted Osmotic Lysis by Simultaneous Blockade of Sodium Pumps and Stimulation of Voltage‐Gated Sodium Channels
Authors: Paul Dennis , Gould Harry J.
Published: November 2023

About 80% metastatic carcinomas (MCs) overexpress voltage‐gated sodium channels (VGSCs) by 50‐fold or more. We hypothesized that electrical stimulation of VGSCs with concomitant blockade of Na+, K+‐ATPase (sodium pumps) would produce a rapid increase in intracellular sodium, causing an influx of water, leading to lysis of MC cells (Targeted Osmotic Lysis, US Patent #8921320). To test this hypothesis in vitro, we cultured highly metastatic MDA‐MB‐231 cells, which overexpress NaV1.5 channels. For single cell experiments, treatment with the sodium pump blocker ouabain and electrical stimulation of the cells produced lysis in as little as 16 sec. Ouabain or electric current alone did not lyse the cells. Treatment did not lyseMCF‐10a or MCF‐12a (normal breast) cells. For experiments in 24‐well plates, entire wells of MDA‐MB‐231 cells were lysed in 10 min. To test the hypothesis in vivo, we established MDA‐MB‐231allographs in Nu/J nude mice. When tumor diameter reached approximately 1 cm, we administered 10 mg/kg ouabain s.c. or saline. Thirty and 60 min later, tumors received 60 sec of 10V direct current delivered through direct contact stimulation. This single‐treatment produced 70% cell lysis in tumors treated with both ouabain and stimulation. Tumors treated with saline alone and saline plus stimulation averaged 10% lysis. Ouabain alone produced 15% cell death. Treatment did not affect non cancerous muscle tissue surrounding the tumor. In survival studies, single day treatment attenuated tumor growth by 45%. Three treatments separated by 3 days attenuated growth by 60–100%. Thus, targeted osmotic lysis is a promising option as an effective treatment of MCs that overexpress VGSCs.

Support or Funding Information

This research was supported by the LSU Depts. of Pharmacology and Neurology, and Oleander Medical Technologies, LLC.
Glucose‐Mediated Regulation of UQCRFS1 and the Cardiac Metabolome
Authors: Williams Lamario J , Brahma Manoja K , McCrory Mark M , Wende Adam R
Published: November 2023

Diabetes, among other metabolic disorders, is associated with harmful changes to the heart. One potential mechanism for this is reduced flexibility in mitochondrial substrate utilization leading to heart failure. The goal of this project is to define the molecular mechanisms by which diabetes contributes to the development of mitochondrial dysfunction. Accumulating evidence suggests that increased flux of glucose through non‐oxidative pathways such as the hexosamine biosynthetic pathway (HBP) may lead to cardiovascular complications in diabetic patients. HBP produces UDP‐N‐acetylglucosamine (UDP‐GlcNAc) that provides the sugar molecule for O‐linked attachment of GlcNAc moieties to specific serine/threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. We hypothesized that hyperglycemia‐induced mitochondrial protein O‐GlcNAcylation may contribute to the impaired substrate utilization resulting in increased reactive oxygen species (ROS) production in the diabetic myocardium. Excess ROS production has been widely implicated in both the onset of diabetes and many of its complications. To test this hypothesis, we used the streptozotocin‐induced diabetic mouse model. In addition, to investigate the glucose‐specific contribution, an inducible transgenic mouse model overexpressing glucose transporter 4 (GLUT4; mG4H) specifically in cardiomyocytes was used. We found that increased flux of glucose leads to enhanced posttranslational modification of mitochondrial proteins by O‐GlcNAcylation in diabetic and mG4H mice. From wheat germ agglutinin enrichment and immunoprecipitation assays, we have identified that UQCRFS1, an OXPHOS complex III protein, is O‐GlcNAcylated in diabetic and mG4H mice heart. In addition, microarray and qPCR analysis confirmed that Uqcrfs1 mRNA levels were unchanged suggesting that this regulation does not involve transcriptional pathways and is specific to O‐GlcNAc of UQCRFS1 in the development of mitochondrial dysfunction. Future goals for this project are to identify diabetic signatures that define cardiac mitochondrial dysfunction related to decrease in OXPHOS complex activity. To test this we have also developed an in vitro model using immortalized human cardiomyocytes that can be tested using the Seahorse Flux Analyzer to directly interrogate each OXPHOS complex. Further characterization of GlcNAc‐mediated regulation of UQCRFS1 in response to increased glucose delivery and substrate overload will be confirmed using immunoprecipitation followed by mass spectrometry to define this mechanism in disease progression in a diabetic myocardium. Together successful completion of these studies will provide novel insights into the molecular mechanisms by which excess glucose in diabetes contributes to mitochondrial dysfunction.

Support or Funding Information

NIH R00 HL111322 and HL111322‐04S1 to A.R.W.

NIH R25 DK078381 to L.J.W.
Regression of pressure overload‐induced cardiac hypertrophy by trientine‐induced copper redistribution to the heart in rats
Authors: Liu Jiaming , Liu Yinjie , Fu Chunyan , Xiao Ying , Feng Qipu , Fan Suzhen , Ding Xueqin , Qiu Liying , Han Pengfei , Kang Y. James
Published: November 2023

Sustained pressure overload leads to pathological cardiac hypertrophy, and copper efflux from the heart with an increase in plasma copper concentrations. Dietary copper supplementation replenishes copper content and reverses the pathogenesis in mice with cardiac hypertrophy. We recently observed that copper‐trientine complex increased copper uptake by primary cultures of neonatal rat cardiomyocytes. The present study was undertaken to test the hypothesis that trientine can redistribute copper from plasma to the copper‐depleted heart tissue. Adult male Sprague‐Dawley rats were subjected to ascending aortic constriction (AAC). Sixteen weeks after the operation, cardiac hypertrophy was observed by echocardiography and trientine at a dose of 8 mg/kg, twice a day, was administered orally for 6 weeks. At the end of the treatment, copper concentrations were significantly increased in the heart and significantly reduced in the plasma. Along with copper redistribution, cardiac hypertrophy was reversed almost back to the normal as judged by IVSD and LVPWD, and cardiac function was improved as indicated by EF and FS. In addition, the level of cardiac fibrosis was reduced as reflected by the decrease in the content of collagens type I and III and the reduced cross linking of the collagens. This study demonstrates that trientine, although traditionally considered as a copper chelator, is capable of redistributing copper from plasma to the heart leading to regression of pathological cardiac hypertrophy and improved cardiac function.

Support or Funding Information

National Science Foundation of China (81230004 and 81300109).
Optical cryo‐imaging reveals a heterogeneous distribution of mitochondrial redox state in the ex‐vivo guinea pig heart and its alteration during ischemia and reperfusion
Authors: Camara Amadou K.S. , Salehpour Fahimeh , Motlagh Mohammad M , Sepehr Reyhaneh , Heisner James S , Dash Ranjan K , Ranji Mahsa
Published: November 2023

Rationale

Oxidation of substrates to generate ATP in mitochondria is mediated through the redox reactions of reducing equivalents, NADH and FADH2, via the mitochondrial electron transport chain. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos), in part, by altering the mitochondrial redox states. We have shown previously using a fiber optic probe online (spectroflurometrically) in the ex‐vivo heart changes in the mitochondrial redox states before, during, and after IR. However, this approach is limited in that it only shows the changes in the redox states in a patch of cardiac tissue in the left ventricular free wall. The heart is a complex and heterogeneous organ anatomically and physiologically, and alteration of the metabolic heterogeneity can portend the state of its health. We hypothesize that IR alters the metabolic heterogeneity of the mitochondrial redox state of the heart that is only evident in the 3‐dimensonal (3‐D) optical cryo‐imaging of the perfused heart before, during, and after IR.

Methods

The study involved four groups of hearts: time control (TC: heart perfusion without IR), perfusion followed by global ischemia only (Isch), perfusion followed by IR, and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online (using a PTI system) in guinea pig ex‐vivo perfused hearts in the Langendorff mode before, during, and after IR. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3‐D optical cryo‐imaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR).

Results

The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. During ischemia, the RR distribution appeared to be similar to the TC and the distribution of the RR in the IR group displayed a shift that is different from the TC group. The spectrofluorometric data of the RR paralleled our previous results in the changes in the redox state during IR. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with control (TC).

Conclusion

The 3‐D optical cryo‐imaging of the heart provides novel insights into the heterogeneous distribution of the mitochondrial RR and metabolism from the base to the apex during ischemia and IR. This 3‐D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data.

Support or Funding Information

The work was supported by the following grants: NIH P01 GM 066730 (ZB PI/PD); NIH R01 HL095122 (RKD and AKSC); CTSI and RGI grants (MR).
Determination of Regional Flow Utilizing Microspheres in ex vivo Myocardial Ischemia/Reperfusion (MI/R) Injury
Authors: Kuhn Tarah F. , Clark Sabrina , Kuo Daniel , Liu Stephanie , Chen Qian , Barsotti Robert , Young Lindon H.
Published: November 2023

MI/R results in marked cardiac contractile dysfunction and cell death. We previously discovered that protein kinase C epsilon peptide inhibitor (PKC ɛ‐) robustly restored postreperfused cardiac function, and reduced infarct size, oxidative stress, and leukocyte endothelial interactions in coronary, hind limb, renal, and mesenteric vascular inflammation models. The mechanisms of these effects in part are due to attenuating uncoupled endothelial nitric oxide (NO) synthase activity and increasing endothelial NO bioavailability. Therefore, we hypothesize that PKC ɛ‐will increase regional flow during reperfusion. We determined regional flow to the right ventricle, left ventricle, and septum at baseline and after 10 and 45 min reperfusion using fluorescent microspheres in isolated perfused rat hearts subjected to global I(30min)/R(45min). A cell permeable PKC ɛ‐(myr‐EAVSLKPT, MW=1054 g/mol, 10μM, n=8) was given at beginning of reperfusion for 5 min. We found that final left ventricular developed pressure (LVDP) recovered to 70 ± 7%, maximal rate of left ventricular contraction, (+dP/dtmax) to 57 ± 7% and maximal rate of left ventricular relaxation, (−dP/dtmin) to 58 ± 6% of baseline values. These parameters were significantly improved compared to untreated control I/R hearts (n=7) that only recovered to 30 ± 5% in LVDP, 21 ± 5% in +dP/dtmax and 25 ± 5% in −dP/dtmin relative to baseline values (all p < 0.01). Moreover, our preliminary data suggest that PKC ɛ‐treatment increased regional flow by 190 ± 40% in the right ventricle, 140 ± 30% in the left ventricle, and 120 ± 20% in the septum at 10 min postreperfusion compared to untreated control I/R hearts. These values were maintained throughout the remaining 35 min of reperfusion. In summary, the data indicates that PKC ɛ‐improves postreperfused cardiac function in part by restoration of regional flow.

Support or Funding Information

This study was supported by the Department of Bio‐Medical Sciences, Division of Research and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine and the Pennsylvania Department of Health.
Does Protein Sulfuration Take Place in the Heart?
Authors: Sonobe Takashi , Haouzi Philippe
Published: November 2023

Endogenous hydrogen sulfide (H2S) has been suggested to play a physiological role through protein sulfuration (also referred as sulfhydration) in the regulation of cardiac contractility. The sulfuration is a process by which H2S combines to free cysteine residue on proteins to create persulfide (protein‐S‐SH), changing protein conformation, such as in calcium channels, and functions. Since any new molecule of free H2S produced will immediately combine with the iron present on myoglobin or will be oxidized, our objective was to determine whether increasing endogenous H2S production can actually increase the pool of H2S combined with cysteine residues in the heart.

To achieve this goal, we infused exogenous H2S up to a level that will alter cardiac contractility in order to create conditions wherein soluble H2S concentrations increase to levels higher than those reached by endogenous sulfide in the heart. The presence of sulfurated protein in the heart was identified by measuring gaseous H2S released after reducing disulfide bonds. TCEP was chosen as a potent reducing agent instead of dithiothreitol (DTT), previously used for this purpose in the literature, as DTT possesses two thiol groups (−SH) a significant source of H2S. Hearts were removed from control animals and animals exposed to 0.8 mg/min NaHS until cardiac depression‐induced hypotension was produced. The hearts were immediately homogenized in glycine‐NaOH buffer (pH 9), to prevent any evaporation of gaseous H2S. The homogenate was transferred into the 50‐ml chamber filled with sodium phosphate buffer (final pH ~6). At this pH, HS− will easily convert into gaseous H2S, in keeping with H2S partial pressure, and measured in the chamber headspace. The headspace was continuously flowed with N2, and H2S was directly measured by a voltammetry H2S analyzer before and after TCEP (5 mM).

We found that the level of H2S produced by TCEP was similar and very low (below 0.5 nmol/g of protein) in both the control and intoxicated hearts, despite levels of free H2S in the blood above 10 microM in the latter. In contrast, the pool of H2S in albumin solution (10% in saline) incubated with H2S for 10 min, increased as a function of H2S concentrations using 0, 2, and 20 microM NaHS and reaching 10.6 ± 1.1, 11.8 ± 0.7 and 13.3 ± 0.4 nmol/g respectively. The change in H2S remained however extremely modest.

Toxic levels of soluble H2S were unable to increase the pool of H2S liberated from the heart in a strong reducing environment. This suggests that free H2S in the heart is immediately trapped by other compounds such metallo‐proteins, e.g. myoglobin, present in the heart or is oxidized in the mitochondria before reacting with proteins, in contrast to a solution of albumin unable to produce these effects. Even in the latter, the pool of H2S produced by an increase in free H2S appears to be extremely small and to require toxic levels of sulfide to be affected.
Effects of excess succinate on cardiomyocyte mitochondrial H2O2 production and cell death
Authors: Williams Anna Marie , Olson Jessica , Yan Yasheng , Bai Xiaowen , Camara Amadou K.S. , Bosnjak Zeljko J.
Published: November 2023

Introduction

During myocardial infarction injury, reactive oxygen species (ROS) generation in the first few minutes of reperfusion has been shown to cause significant cellular dysfunction and death of the myocardium. Recent studies suggest ROS generation during reperfusion is due to accumulation of intermediate citric acid cycle metabolites, specifically succinate accumulation. Additionally, previous studies suggest that an increase in fumarate accompanied by an increase in succinate is cardioprotective. In this study, we examined the effect of excess succinate on H2O2 production and cell death. We hypothesized that excess succinate increases H2O2 production through reverse electron transport in mitochondria isolated from Sprague‐Dawley rat hearts and increases cell death in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) and that addition of fumarate would abolish this phenotype.

Methods

The left ventricles of Sprague Dawley male rats were collected and minced. Mitochondria were isolated using differential centrifugation. Mitochondria were exposed to 5 mM succinate with or without 1μM rotenone (a complex I inhibitor). H2O2 production was measured using an Amplex Red assay. Human iPSCs were cultured to confluency and differentiated into CMs. Contracting cells were immunostained for cardiac specific markers to confirm >90% purity of CMs. CMs were exposed to 5 mM dimethyl succinate with or without 5 mM dimethyl fumarate for 1 hour followed by 24 hours of reperfusion in basal media. Cell viability was measured using propidium iodide staining.

Results

Excess succinate caused a significant increase in H2O2 production in mitochondria isolated from Sprague Dawley rat hearts. This effect was abolished with addition of rotenone, showing that H2O2 production was occurring through reverse electron transport. Dimethyl succinate also caused significant cell death in iPSC‐derived CMs. Dimethyl fumarate with dimethyl succinate did not abolish increased cell death.

Conclusions

We have confirmed that excess succinate causes a significant increase in H2O2 production through reverse electron transport in mitochondria isolated from Sprague Dawley rat hearts and a significant increase in cell death in iPSC‐derived CMs. Fumarate in addition to excess succinate did not have a cardioprotective effect in iPSC‐CMs. However, this may be a toxic effect rather than a metabolic effect. Further experiments detailing the mechanism by which this occurs may reveal new therapeutic targets to reduce the detriment of ROS production from ischemia/reperfusion injury.

Support or Funding Information

NIH/NIGMS Program Project Grant 2P01GM066730‐11Program Director: Zeljko J. Bosnjak “Mechanisms of Anesthetic Cardioprotection”Project II: “MicroRNA in Anesthetic Cardioprotection”