Tyrode's Solution Research Articles

Arrhythmogenic Potential of Myocardial Edema: The Interstitial Osmolality Induces Spiral Waves and Multiple Excitation Wavelets.

Myocardial edema is a common symptom of pathological processes in the heart, causing aggravation of cardiovascular diseases and leading to irreversible myocardial remodeling. Patient-based studies show that myocardial edema is associated with arrhythmias. Currently, there are no studies that have examined how edema may influence changes in calcium dynamics in the functional syncytium. We performed optical mapping of calcium dynamics on a monolayer of neonatal rat cardiomyocytes with Fluo-4. The osmolality of the solutions was adjusted using the NaCl content. The initial Tyrode solution contained 140 mM NaCl (1T) and the hypoosmotic solutions contained 105 (0.75T) and 70 mM NaCl (0.5T). This study demonstrated a sharp decrease in the calcium wave propagation speed with a decrease in the solution osmolality. The successive decrease in osmolality also showed a transition from a normal wavefront to spiral wave and multiple wavelets of excitation with wave break. Our study demonstrated that, in a cellular model, hypoosmolality and, as a consequence, myocardial edema, could potentially lead to fatal ventricular arrhythmias, which to our knowledge has not been studied before. At 0.75T spiral waves appeared, whereas multiple wavelets of excitation occurred in 0.5T, which had not been recorded previously in a two-dimensional monolayer under conditions of cell edema without changes in the pacing protocol.

Unlocking the Potential of Assisted Hatching in Assisted Reproductive Technology: A Narrative Review.

The present study is set in the broad field of assisted reproductive technologies (ARTs) and examines various procedures under assisted hatching (AH). It also reviews their effects on implantation success rates. The primary emphasis is on explaining who has benefited and how many have benefited from these interventions. The most important factor determining the success rate of ART is implantation. To increase these rates, we use AH in our clinics to enhance each embryo's chances at life and substantially improve overall results. This comprehensive review includes various approaches, such as chemical-based measures (such as applying Tyrode's solution) and mechanical techniques (such as zona drilling and partial zona dissection). The individual techniques are carefully scrutinized, considering their mechanical detailing, methods of applying therapeutic effects, and the appropriateness of matching present social circumstances. The review begins by analyzing the basic nature of AH as a medium for embryo implantation and then focuses on how this detailed view reveals the advantages and drawbacks of various methods. Moreover, the articles discuss improvements in AH technology and many of the most modern technological developments that can help fine-tune ART issues. A major problem with these methodologies is that they involve serious risks and legal complications. However, a broad assessment of these topics allows us to understand their impact on fertility treatments. This review is written as a guidebook for physicians and researchers working in reproductive medicine. It compiles all current knowledge, providing literature to build on successes that will make breakthroughs possible in ART. Indeed, this is a valuable reference for guiding us in navigating complex AH procedures. It advances ARTs step by step toward perfection.

Electrochemical dissolution and retrieval of broken NiTi endodontic files from root canal using chloride based isotonic fluids as electrolytes - An in vitro study.

The electrochemical dissolution method of instrument retrieval emphasizes on the dissolution of the instrument rather than sacrificing dentine. Most of the studies conducted for electrochemical dissolution used fluoride-containing electrolytes and were performed inside a beaker. In this study, we used chloride-based fluids as electrolytes. Fifty extracted mandibular first premolars were divided into five groups based on the electrolytes used. Canals were enlarged to ProTaper Universal F2, and files were intentionally broken inside the canal. These specimens were subjected to electrochemical characterization by applying the potential of 9V for 20 min. Optical images were taken to assess the change in surface topography. The results were analyzed statistically by one-way analysis of variance (analysis of variance [ANOVA]). The rate of dissolution based on the electrolyte used decreased in the following order, viz. Tyrode's solution>artificial saliva>normal saline>Ringer's lactate/physiological serum. Apart from fluoride, chloride-based electrolytes could be an efficient alternative.

The effect of zinc on the stiffness of chicken lenses

Aims/Purpose: Aquaporins are specialized water channels. A volume change through permeable aquaporins may be necessary for the lens' ability to change shape during accommodation1. This study aimed to determine the effects of zinc2, an agonist that increases aquaporin 0 (AQP0) permeability in oocytes, on the biomechanics of chicken lenses.Methods: White Leghorn chickens (Gallus gallus domesticus, N = 6) were sacrificed at 14 days of age. Both eyes were enucleated and lenses were dissected in Tyrode's solution (TS). One lens from each bird was treated with zinc chloride (80 μg/mL in TS for 15 min, at room temperature; RT), while the other lens (control lens) was incubated in TS (15 min, RT). Lenses were compressed (CellScale Micro Tester G2) to test their biomechanical integrity. For each lens, force‐compression data were generated by the MicroTester G2 software (ver. 1.0.0.1) and fit to a three‐parameter exponential curve with the equation y = y0 + aebx; the unitless b‐coefficient of the exponential equation was used to assess stiffness as it describes how rapidly the force increases as the compression distance increases. B‐coefficients from each curve were extracted and the effects of zinc treated versus control lenses were analysed using a paired t‐test.Results: The b‐coefficient value for zinc‐treated lenses averaged to 4.2 ± 0.8 while the mean value for control lenses was significantly (p = 0.01) lower at 3.2 ± 0.5, indicating that zinc‐treated lenses were stiffer.Conclusions: The compression data show a zinc‐associated increase in lens stiffness. AQP0 typically plays a role in allowing water to enter, but not exit, the lens, therefore zinc may have led to an increase in water content, resulting in stiffer lenses. However, water permeability was not measured in this study, therefore the AQP0‐associated mechanism of lens stiffening cannot be confirmed. Further study is needed to determine zinc's action on the biomechanics of the lens.

Abstract 18867: A Novel Light-Sensitive Ex-Vivo Rat Atrial Anatomical Model for Studying Atrial Arrhythmias and Cardioversion

Introduction: Atrial arrhythmias, including atrial fibrillation (AF), contribute to significant morbidity and mortality. However, the lack of suitable tissue models in small animals hampers our understanding of the underlying mechanisms. Optogenetic actuators offer precise and reversible stimulation with high spatiotemporal resolution, presenting a unique opportunity to investigate atrial arrhythmias. Hypothesis: Our aim was to develop a light-sensitive ex-vivo anatomical model in rats for studying atrial arrhythmias and to utilize this model for optimizing optogenetic stimulation and cardioversion strategies. Methods: We injected purified AAV particles containing the transgenic light-sensitive channel (CoChR) into 5-day-old Wistar rats, followed by an 8-10 week period for atrial growth. The atria were then isolated, flattened, and affixed to a custom-made silicon plate. Superfusion with oxygenated Tyrode's solution and loading with Di-4-ANBDQBS allowed for high-resolution optical mapping. Atrial arrhythmias were induced through targeted tachypacing combined with carbamylcholine treatment. Additionally, we compared different optical and pharmacological treatment strategies to evaluate their efficacy in modulating atrial arrhythmias. Results: Immunostaining confirmed atrial expression of CoChR. Optical mapping revealed a functional syncytium with sinus node activation and propagation throughout the atria. Pacing at increasing frequencies resulted in a relatively flattened action potential duration restitution curve. Successful induction of reentrant arrhythmias was achieved. Additionally, we demonstrated the feasibility of optogenetic actuators for atrial pacing, modulation of spontaneous activity, and optical termination of arrhythmias. Conclusions: The developed optogenetic superfused atria model provides a valuable platform for studying atrial electrophysiology and modeling reentrant arrhythmias. Furthermore, it enables the optimization of optogenetic stimulation and cardioversion strategies. This integrated approach enhances our understanding of atrial arrhythmias and holds promise for the development of novel therapeutic interventions.

The ethanol extract of Euphorbia hirta exhibits in vitro antidiabetic potentials by sensitization of the insulin

The objective of current investigation was to assess the mechanism of action of Euphorbia hirta for its antidiabetic properties. Materials &Methods: The authenticated leaves of plant Euphorbia hirta were dried and powdered. The powdered drug was defatted with petroleum ether and subjected to ethanol extraction. The ethanol extract of Euphorbia hirta was subjected to preliminary phytochemical investigation. The glucose up takes by rat hemi-diaphragm (skeletal muscle) model was also used evaluate potentials of the ethanol extract of Euphorbia hirta to enhance utilization of the blood glucose by peripheral tissues. The overnight fasted albino rat was sacrificed and dissected to hemidiaphragm. Weighed quantity of skeletal muscle was incubated with glucose in Tyrode solution for 30 mins at 37oC and the amount of glucose utilized by the tissue was determined. Results: The ethanol extract of Euphorbia hirta significantly increased the glucose utilization by skeletal muscle which its shows potency to sensitize the insulin. Conclusion: The results of the presents investigation recommends that, one of the mechanisms of ethanol extract of Euphorbia hirta for antidiabetic activity is reduction of insulin resistance.

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone.

Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP-amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570-595 nm for the cytosolic Ca2+ and 610-750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. The CTP-amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP-amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP-amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP-amiodarone significantly decreased the calcium transients compared to the controls. CTP-amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1β, and IL-6. Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

Effect of Dihydroartemisinin on contractility to Drugs in isolated smooth muscle Preparations in guinea pig in vitro models

This study was aimed at evaluating the effect of Dihydroartemisinin (DHA)on contractility to drugs in ileum isolated smooth muscle preparations in guinea pigs using in Vitro experimental procedures / standard protocols in an organ bath set up. dihydroartemisinin (1.0 x 10 - 8 - 1.0 x 10 – 5mg/mL) when applied alone and separately excited marked variable effects on guinea pig ileum . In some preparation it showed no response, while in others it produced slight phasic contraction when external calcium (Ca2+) ion was introduced. The slight phasic contractile activity was abolished by verapamil (5x10-3mg/mL). Dihydroartemisinin (1.0 x 10 - 6 - 1.0 x 10 – 5mg/mL) caused marked significant induced contraction – dependent inhibition of acetylcholine, potassium chloride and histamine in depolarizing tyrode solution (p &lt; 0.05).The Inhibitory response maxima of dihydroartemisinin on acetylcholine induced contraction is 17.0±0.1 mm(65.56% inhibition) ; and is moderate when compared to the inhibitory effect of atropine (10-5 M). The result shows that dihydroartemisinin seems to act via non-specific receptor mechanism, with appreciable calcium channel blocking activity and it is safe at therapeutic doses.

Kir1.1 and SUR1 are not implicated as subunits of an adenosine triphosphate-sensitive potassium channel involved in diazoxide cardioprotection

ObjectiveThe adenosine triphosphate-sensitive potassium channel opener diazoxide mimics ischemic preconditioning and is cardioprotective. Clarification of diazoxide's site and mechanism of action could lead to targeted pharmacologic therapies for patients undergoing cardiac surgery. Several mitochondrial candidate proteins have been investigated as potential adenosine triphosphate-sensitive potassium channel components. Renal outer medullary potassium (Kir1.1) and sulfonylurea sensitive regulatory subunit 1 have been suggested as subunits of a mitochondrial adenosine triphosphate-sensitive potassium channel. We hypothesized that pharmacologic blockade or genetic deletion (knockout) of renal outer medullary potassium and sensitive regulatory subunit 1 would result in loss of diazoxide cardioprotection in models of global ischemia with cardioplegia. MethodsMyocyte volume and contractility were compared after Tyrode's physiologic solution (20 minutes), stress (hyperkalemic cardioplegia ± diazoxide, ± VU591 (Kir1.1 inhibitor), N = 9 to 23 each, 20 min), and Tyrode's (20 minutes). Isolated mouse (wild-type, sensitive regulatory subunit 1 [−/−], and cardiac knockout renal outer medullary potassium) hearts were given cardioplegia ± diazoxide (N = 9-16 each) before global ischemia (90 minutes) and 30 minutes reperfusion. Left ventricular pressures were compared before and after ischemia. ResultsStress (cardioplegia) was associated with reduced myocyte contractility that was prevented by diazoxide. Isolated myocytes were not responsive to diazoxide in the presence of VU591. In isolated hearts, diazoxide improved left ventricular function after prolonged ischemia compared with cardioplegia alone in wild-type and knockout (sensitive regulatory subunit 1 [−/−] and cardiac knockout renal outer medullary potassium) mice. ConclusionsIsolated myocyte and heart models may measure independent and separate actions of diazoxide. By definitive genetic deletion, these data indicate that sensitive regulatory subunit 1 and renal outer medullary potassium are not implicated in cardioprotection by diazoxide.

FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.

Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated. 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting. Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2. Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.

Role of the succinate and GPR91 pathway in atrial fibrillation mechanisms

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity and mortality. The mechanisms underlying the transition from paroxysmal to persistent AF are still a matter of debate. Recent studies indicate a potential role for metabolic remodelling in the AF stabilization process and increased levels of a specific metabolic substrate, succinate, have been found in blood plasma of AF patients. While succinate is known to increase reactive oxygen species (ROS) production, the role of the succinate receptor (GPR91) in atrial function is unknown. To assess the impact of the succinate pathway on AF vulnerability in a sheep model. GPR91 expression levels were quantified by Western Blot (WB) in a previously described sheep model of persistent AF induced by burst-pacing. Optical experiments were subsequently performed in ex vivo right atria (RA) from control sheep (n = 5). RA were perfused by Tyrode solution, then supplemented with the cis-epoxysuccinic acid (cESA), a specific activator of GPR91 (150 μM) to study its involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5 Hz pacing frequency. We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30 Hz) to assess ex vivo AF vulnerability. Finally, we investigated these properties in a human RA from an AF patient. WB experiments revealed an increase in GPR91 expression in AF sheep compared to sham sheep. Interestingly, in resistant sheep (no AF after 90 days of burst pacing), expression levels were decreased compared to sham. During cESA perfusion in control sheep RA, sinus rhythm (119 vs. 95 bpm; P = 0.03) and ERP (202 vs. 178 ms; P = 0.36) were decreased when compared to baseline. We also observed a decrease in APD80 (199 vs. 177 ms; P = 0.009) and in CV (127 vs. 115 cm/sec; P = 0.01). These electrophysiological perturbations led to an increase in AF vulnerability by burst pacing (0% vs. 60%) and an increase in spontaneous arrhythmias. These results were corroborated in the human RA were cESA decreased APD80 (265 vs. 241 ms), ERP (240 vs. 220 ms), and CV (119 vs. 94 cm/sec) and increased arrhythmia vulnerability. For the first time, we demonstrated that GPR91 is expressed in atrial tissue and that its activation through succinate can play a major role in AF mechanisms.

PO-01-159 ROLE OF THE SUCCINATE PATHWAY IN ATRIAL FIBRILLATION MECHANISMS

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity and mortality. The mechanisms underlying the transition from paroxysmal to persistent AF are still a matter of debate. Recent studies indicate a potential role for metabolic remodelling in the AF stabilization process and increased levels of a specific metabolic substrate, succinate, have been found in blood plasma of AF patients. While succinate is known to increase reactive oxygen species (ROS) production, the role of the succinate receptor (GPR91) in atrial function is unknown. To assess the impact of the succinate pathway on AF vulnerability in a sheep model. GPR91 expression levels were quantified by Western Blot (WB) in a previously described sheep model of persistent AF induced by burst-pacing. Optical experiments were subsequently performed in ex vivo right atria (RA) from control sheep (N=5). RA were perfused by Tyrode solution, then supplemented with the cis-epoxysuccinic acid (cESA), a specific activator of GPR91 (150μM) to study its involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5Hz pacing frequency. We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30Hz) to assess ex vivo AF vulnerability. Finally, an organ donation program allowed us to investigate these properties in a human RA from an AF patient. WB experiments revealed an increase in GPR91 expression in AF sheep compared to sham sheep. Interestingly, in resistant sheep (no AF after 90 days of burst pacing), expression levels were decreased compared to sham. During cESA perfusion in control sheep RA, sinus rhythm (119 vs 95 bpm; p=0,03) and ERP (202 vs 178 ms; p=0,36) were decreased when compared to baseline. We also observed a decrease in APD80 (199 vs 177 ms; p=0.009) and in CV (127 vs 115 cm/sec; p=0.01). These electrophysiological perturbations led to an increase in AF vulnerability by burst pacing (0% vs 60%) and an increase in spontaneous arrhythmias. These results were corroborated in the human RA were cESA decreased APD80 (265 vs 241 ms), ERP (240 vs 220 ms), and CV (119 vs 94 cm/sec) and increased arrhythmia vulnerability. For the first time, we demonstrated that GPR91 is expressed in atrial tissue and that its activation through succinate can play a major role in AF mechanisms.

Acellular hemoglobin impairs calcium release and contractility in cardiomyocytes from healthy animals

Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOC) have been proposed as an alternative for blood transfusion. Phase III clinical trials of previous HBOC generations presented issues, such as vasoconstriction and myocardial infarction, that were not detected in pre-clinical trials. These problems have been attributed to extravasation of the low molecular weight (LMW) Hb species, nitric oxide (NO) scavenging and O 2 oversupply to the vasculature. However, the mechanisms responsible for the cardiac toxicity of HBOCs remain unclear. To explore the effects of acellular (acell) Hb and HBOC in cardiac tissues, we exposed cardiomyocytes (CMCs) to highly purified stroma-free acell bovine Hb (bHb) or human Hb (hHb). CMCs were loaded with Ca2+ fluorescent indicator (fluo-3) and put on laminin-coated coverslips. Cells were superfused with standard Tyrode solution with and without acell Hb. CMCs were stimulated with current pulses given at 1 Hz, and free cytosolic Ca2+ concentration was measured. To eliminate the effects of NO scavenging on CMCs, L-Name was added to the superfused with bHb and hHb. To study the effect of acell Hb MW on CMCs, LMW polymerized hHb (PolyhHb) was used to challenge CMCs. To explore the effect of Hb oxidation on CMCs, LMW MetHb was used to challenge the CMCs and compared to LMW PolyhMetHb. To explore the effects of antioxidants and heme toxicity, the studies with hHb and MethHb were repeated in the presence of the antioxidant N-acetylcysteine (NAC) or purified human Haptoglobin (Hp). Mitochondrial reactive oxygen species (ROS) production was measured using the fluorescent indicator MitoSOX Red after removing the Hb by washing the cells. After ROS determination, the maximal ROS production by CMCs was measured by challenging the cells with a high concentration of H 2 O 2 . Acell Hb increased the Ca 2+ transient decay time constant (tau) relative to control. Removing the effects of NO production with L-Name, increased the tau of the Ca 2+ transient in control and acell Hb challenge cells. Challenging CMCs with PolyhHb reduced the effects observed with acell Hb in Ca 2+ transients. MetHb appears to be worse than reduced Hb in terms of the changes induced to CMCs Ca 2+ transients. The antioxidant NAC mitigated the effects of acell Hb and MetHb but did not eliminate the changes on increased Ca2+ transients. The Hb scavenger, Hp, mitigated the effects of acell hHb and MethHb on Ca 2+ transients. The MitoSOX Red fluorescence was higher for CMCs exposed to acell Hb and MetHb compared to the control. PolyhHb and PolyMetHb challenge decreased the MitoSOX compared to acell hHb and MethHb. Cells with higher levels of ROS showed lower changes in fluorescence when exposed to H 2 O 2 . Here, we show that the toxicity of acell Hb is impart determined by facilitated diffusion, NO scavenging, heme release, and ROS, potentially affecting the energy supply and metabolism of CMCs. This study was supported by National Institutes of Health grants R01HL162120, R01HL159862 and the Department of Defense under grants W81XWH1810059. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

Reduction of intracellular Mg2+ caused by reactive oxygen species in rat ventricular myocytes.

The concentration of intracellular free Mg2+ ([Mg2+]i) should be maintained strictly for the regulation of cellular functions. Since reactive oxygen species (ROS) are liable to increase in various pathological conditions and induce cellular damage, we investigated whether ROS affect intracellular Mg2+ homeostasis. We measured [Mg2+]i in ventricular myocytes from Wistar rats using the fluorescent indicator, mag-fura-2. The administration of hydrogen peroxide (H2O2) decreased [Mg2+]i in Ca2+-free Tyrode's solution. Intracellular free Mg2+ was also reduced by endogenous ROS as generated by pyocyanin, which was inhibited by pretreatment with n-acetyl cysteine (NAC). The rate of change in [Mg2+]i by 500 μM H2O2 in 5 min (on average, -0.61 μM/s) was independent of extracellular Na+, and intra- and extracellular Mg2+ concentrations. When extracellular Ca2+ was present, the rate of Mg2+ decrease was significantly reduced, on average, by ∼60%. The half-maximal effective concentration of H2O2 on the Mg2+ decrease was estimated to be between 400 and 425 μM. The Mg2+ decrease by H2O2 in the absence of Na+ was inhibited by 200 μM imipramine, a known inhibitor of Na+/Mg2+ exchange. We perfused rat hearts with the Ca2+-free Tyrode's solution containing H2O2 (500 μM, 5 min) on the Langendorff apparatus,. H2O2 stimulation increased Mg2+ concentration in the perfusate, suggesting the H2O2-induced decrease in [Mg2+]i was caused by Mg2+ extrusion. Collectively, these results suggest the existence of a Na+-independent Mg2+ efflux system activated by ROS in cardiomyocytes. The lower [Mg2+]i may in part be attributed to ROS-mediated cardiac dysfunction.

Mg2+ extrusion activated by hydrogen peroxide in rat ventricular myocytes.

To investigate the effect of reactive oxygen species (ROS) on intracellular-free Mg2+ concentration ([Mg2+]i), we measured [Mg2+]i in rat ventricular myocytes using a fluorescent indicator, mag-fura-2. The treatment of hydrogen peroxide (H2O2, 500 μM) for 5 min decreased [Mg2+]i from 1.02 ± 0.08 (mean ±SD, n = 7) to 0.75 ± 0.18 mM (p = 0.018, by Wilcoxon signed-rank test) in ∼9 min (including the 5-min treatment and subsequent washout). [Mg2+]i was continuously decreased in ∼ 20 min, and then recovered to the basal level (∼1.0 mM) gradually in the Ca2+-free Tyrode's solution. The half maximal effective concentration of H2O2 on the Mg2+- decrease was estimated to be between 400 and 425 μM. The rate of decrease in [Mg2+]i by H2O2 in the Ca2+-free Tyrode's solution (−0.61± 0.42 μM/s, n = 36) was not significantly different from that obtained in the absence of extracellular Na+ or Mg2+. The addition of 1 mM Ca2+ extracellularly slowed the rate of Mg2+- decrease, on average, by ∼60% (p = 0.002, by Mann-Whitney U-test). The H2O2-induced Mg2+ decrease in Na+-free (substituted with N-methyl-d-glucamine) /Ca2+-free Tyrode's solution was inhibited by 200 μM imipramine, a known inhibitor of Na+/Mg2+ exchange. Rat hearts were perfused with the Ca2+-free Tyrode's solution containing 500 μM H2O2 for 5 min on the Langendorff apparatus. The Mg2+-concentration of the cardiac perfusate was significantly increased after H2O2 administration, suggesting that the H2O2-induced Mg2+-decrease was caused by Mg2+- extrusion. Pyocyanin, a generator of reactive oxygen species also reduced [Mg2+]i. The Mg2+-decrease by pyocyanin was inhibited by a pretreatment of n-acetyl cysteine, an anti-oxidant. In conclusion, it is suggested ROS disrupt intracellular Mg2+ homeostasis, and that the existence of a Na+-independent Mg2+- extrusion system activated by H2O2 in cardiomyocytes.

Empagliflozin prevents oxidative stress induced by acute hyperglycemia in cardiac myocytes.

Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, has shown to improve cardiovascular outcomes in patients with diabetes and heart failure. Empagliflozin has also been shown to provide advantageous effects in excised hearts and isolated cardiomyocytes despite SGLT-2 expression lacking in cardiac myocytes. This suggests empagliflozin may have important off-target effects. Previous studies suggested that empagliflozin effects may include attenuation of both reactive oxygen species (ROS) and Ca/calmodulin-dependent kinase II activation, but exact signaling details remained unclear. Using transgenic mice expressing glutaredoxin 1-coupled redox-sensitive green fluorescent protein 2 (Grx1-roGFP2) sensor localized to either the cytosol or mitochondria, we tested for empagliflozin effect on oxidative stress in myocytes bathed in high-glucose (30 mmol/L) containing Tyrode's solution. To assess the changes in redox potential, we exposed each cell to H2O2 and dithiothreitol to calibrate the sensor. We found acute hyperglycemia induced significant increases in ROS over a 3-5min of perfusion, specifically in the cytosolic compartment. This is in line with previous report showing that hyperglycemia activates CaMKII by intracellular O-GlcNAcylation, then the active CaMKII promotes cytosolic ROS production via NAPDH oxidase 2 (NOX2). Importantly, cells preincubated with empagliflozin were fully protected against the hyperglycemia-induced increase in ROS. We also extended our studies on angiotensin II (AngII)-dependent ROS production because AngII activates NOX2, and the excess ROS promotes oxidation- dependent CaMKII activation. Myocytes pretreated with the selective CaMKII inhibitor autocamtide-2-relative inhibitory peptide (cell-permeable myristoylated form) were used to test the role of CaMKII in ROS production by either hyperglycemia or Ang-II. These studies could reveal whether empagliflozin acts on glucose uptake, CaMKII activation, or via direct antioxidant mechanisms. Our findings may provide a mechanistic basis for the positive effects of empagliflozin in myocytes under conditions relevant to heart disease.

Biomimetic calcium phosphate coating on medical grade stainless steel improves surface properties and serves as a drug carrier for orthodontic applications.

Recently, stainless steel (SSL) miniscrew implants have been used in orthodontic clinics as temporary anchorage devices. Although they have excellent physical properties, their biocompatibility is relatively poor. Previously, our group developed a two-phase biomimetic calcium phosphate (BioCaP) coating that can significantly improve the biocompatibility of medical devices. This study aimed to improve the biocompatibility of SSL by coating SSL surface with the BioCaP coating. Titanium (Ti) discs and SSL discs (diameter: 5mm, thickness: 1mm) were used in this study. To form an amorphous layer, the Ti discs were immersed in a biomimetic modified Tyrode solution (BMT) for 24h. The SSL discs were immersed in the same solution for 0h, 12h, 24h, 36h and 48h. To form a crystalline layer, the discs were then immersed in a supersaturated calcium phosphate solution (CPS) for 48h. The surface properties of the BioCaP coatings were analysed. In addition, bovine serum albumin (BSA) was incorporated into the crystalline layer during biomimetic mineralisation as a model protein. The morphology, chemical composition and drug loading capacity of the BioCaP coating on smooth SSL were confirmed. This coating improved roughness and wettability of SSL surface. In vitro, with the extension of BMT coating period, the cell seeding efficiency, cell spreading area and cell proliferation on the BioCaP coating were increased. These in vitro results show that the BioCaP coating can improve surface properties of smooth medical grade SSL and serve as a carrier system for bioactive agents.

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology.

All-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications. We introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. The system uses oblique transillumination, low-cost cameras, light-emitting diodes, and off-the-shelf components (total ) to capture voltage, calcium, and mechanical waves under electrical or optical stimulation. Our results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (chanelrhodopsin2) only under very low green light ( ). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrode's solution at room temperature. As multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

Programmed Death Ligand 1 Is Released in Platelet-Derived Extracellular Vesicles

Background: Programmed death ligand 1 (PD-L1) is expressed on various cell types, including platelets. The PD-L1/PD-1 pathway plays a central role in maintenance of immunologic homeostasis. Platelets express PD-L1 at low levels in healthy individuals, and upregulate PD-L1 in cancer. Platelet PD-L1 has been suggested to reflect intratumoral PD-L1 and predict response to immune checkpoint inhibitor (ICI) therapy. We hypothesized that platelet-derived EVs contain PD-L1 and may be involved in immunomodulatory and/or adverse effects of ICI. Methods: Whole blood was collected in 3.2% sodium citrate from healthy subjects. PRP was isolated by centrifugation, diluted 1:1 in Tyrode's Solution, and platelets isolated by centrifugation at 1400 x g for 5 minutes. The platelet pellet was resuspended in Tyrode's and stimulated by incubation with 10 uM ADP or 1U/mL thrombin. Platelets were removed by centrifugation, and supernatants serially centrifuged at 16,000 x g for 40 min, and at 100,000 x G for two hours to obtain larger EV (e.g. microparticles) and smaller EV (e.g. exosomes). Large and small EV fractions, and pellets from platelets before and after stimulation were solubilized and PD-L1 content determined by immunoblotting using anti-human PD-L1 antibodies. The concentration of platelet-derived EV was also measured using a ZetaView NTA PMX-120. Results: PD-L1 was detected in lysates of unstimulated platelets but reduced after stimulation. PD-L1 was present primarily in the small EV fraction (exosomes), with greater amounts detected after platelet stimulation (Figure). Platelet EV counts increased substantially after platelet stimulation with ADP (120 x 108/ml), or thrombin (74 x 108/ml) compared to unstimulated platelets (31 x 108/ml). Conclusion: These pilot studies suggest that platelet activation results in release of EV containing PD-L1. Studies are in progress to determine whether these EV may regulate responses or toxicities of ICI through transfer of PD-L1 to other cell types, or other mechanisms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal