Subcellular activation of ß-adrenergic receptors using a spatially restricted antagonist.

Gprotein-coupled receptors (GPCRs) regulate several physiological and pathological processes and represent the target of approximately 30% of Food and Drug Administration-approved drugs. GPCR-mediated signaling was thought to occur exclusively at the plasma membrane. However, recent studies have unveiled their presence and function at subcellular membrane compartments. There is a growing interest in studying compartmentalized signaling of GPCRs. This requires development of tools to separate GPCR signaling at the plasma membrane from the ones initiated at intracellular compartments. We leveraged the structural and pharmacological information available for ß-adrenergic receptors (ßARs) and focused on ß1AR as exemplary GPCR that functions at subcellular compartments, and rationally designed spatially restricted antagonists. We generated a cell-impermeable ßAR antagonist by conjugating a suitable pharmacophore to a sulfonate-containing fluorophore. This cell-impermeable antagonist only inhibited ß1AR on the plasma membrane. In contrast, a cell-permeable ßAR antagonist containing a nonsulfonated fluorophore efficiently inhibited both the plasma membrane and Golgi pools of ß1ARs. Furthermore, the cell-impermeable antagonist selectively inhibited the phosphorylation of PKA downstream effectors near the plasma membrane, which regulate sarcoplasmic reticulum (SR) Ca2+ release in adult cardiomyocytes, while the ß1AR Golgi pool remained active. Our tools offer promising avenues for investigating compartmentalized ßAR signaling in various contexts, potentially advancing our understanding of ßAR-mediated cellular responses in health and disease. They also offer a general strategy to study compartmentalized signaling for other GPCRs in various biological systems.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

Caveolin-3 prevents swelling-induced membrane damage via regulation of ICl,swell activity.

Caveola membrane structures harbor mechanosensitive chloride channels (MCCs; including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current (ICl,swell) and play an important role in cell volume regulation and mechanoelectrical signal transduction. However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells; the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the ICl,swell-selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 µM). These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of ICl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-ß-cyclodextrin. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

Real-World Emission Characteristics of Environmentally Persistent Free Radicals in PM2.5 from Residential Solid Fuel Combustion.

Environmentally persistent free radicals (EPFRs) can induce reactive oxygen species, causing adverse health impacts, and residential fuel (biomass and coal) combustion is believed to be an important emission source for EPFRs; however, the residential emission characteristics of EPFRs are rarely studied in the real world. Here, we conducted a field campaign evaluating the presence and characteristics of EPFRs generated from residential biomass and coal burning in rural China. The emission factors (EFs) of EPFRs (with units of 1020 spins·kg-1) in PM2.5 from the combustion of crop residues (3.97 ± 0.47) were significantly higher than those from firewood (2.06 ± 0.19) and coal (2.13 ± 0.33) (p < 0.05). The EPFRs from residential solid fuel combustion were carbon-centered free radicals adjacent to oxygen atoms. The fuel type was a primary factor controlling EPFR discharge, explaining 68% of the variation in EPFR EFs. The emissions from biomass burning had higher EPFRs per particle than those from coal combustion. EPFRs had stronger relationships with carbonaceous components than with other incomplete combustion products. The EPFRs from biomass burning were mostly generated during the pyrolysis of fuels, while the EPFRs generated from coal combustion were mainly associated with refractory organic compounds. This study provides valuable information for evaluating the fates of EPFRs, promoting a better understanding of the health impacts of air pollution.

Human iPSC-engineered cardiac tissue platform faithfully models important cardiac physiology.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) may provide an important bridge between animal models and the intact human myocardium. Fulfilling this potential is hampered by their relative immaturity, leading to poor physiological responsiveness. hiPSC-CMs grown in traditional two-dimensional (2D) culture lack a t-tubular system, have only rudimentary intracellular calcium-handling systems, express predominantly embryonic sarcomeric protein isoforms, and preferentially use glucose as an energy substrate. Culturing hiPSC-CM in a variety of three-dimensional (3D) environments and the addition of nutritional, pharmacological, and electromechanical stimuli have proven, to various degrees, to be beneficial for maturation. We present a detailed assessment of a novel model in which hiPSC-CMs and hiPSC-derived cardiac fibroblasts are cocultured in a 3D fibrin matrix to form engineered cardiac tissue constructs (hiPSC-ECTs). The hiPSC-ECTs are responsive to physiological stimuli, including stretch, frequency, and ß-adrenergic stimulation, develop a t-tubular system, and demonstrate calcium-handling and contractile kinetics that compare favorably with ventricular human myocardium. Furthermore, transcript levels of various genes involved in calcium-handling and contraction are increased. These markers of maturation become more robust over a relatively short period of time in culture (6 wk vs. 2 wk in hiPSC-ECTs). A comparison of the hiPSC-ECT molecular and performance variables with those of human cardiac tissue and other available engineered tissue platforms is provided to aid selection of the most appropriate platform for the research question at hand. Important and noteworthy aspects of this human cardiac model system are its reliance on "off-the-shelf" equipment, ability to provide detailed physiological performance data, and the ability to achieve a relatively mature cardiac physiology without additional nutritional, pharmacological, and electromechanical stimuli that may elicit unintended effects on function.NEW & NOTEWORTHY This study seeks to provide an in-depth assessment of contractile performance of human iPSC-derived cardiomyocytes cultured together with fibroblasts in a 3-dimensional-engineered tissue and compares performance both over time as cells mature, and with corresponding measures found in the literature using alternative 3D culture configurations. The suitability of 3D-engineered human cardiac tissues to model cardiac function is emphasized, and data provided to assist in the selection of the most appropriate configuration based on the target application.

Epigenetic Priming of Human Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Accelerates Cardiomyocyte Maturation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Can the properties of engineered nanoparticles be indicative of their functions and effects in plants?

The extensive applicability of engineered nanoparticles (ENPs) in various fields such as environment, agriculture, medicine or biotechnology has mostly been attributed to their better physicochemical properties as compared with conventional bulk materials. However, functions and biological effects of ENPs change across different scenarios which impede the progress in their risk assessment and safety management. This review thus intends to figure out whether properties of ENPs can be indicators of their behavior through summarizing and analyzing the available literature and knowledge. The studies have indicated that size, shape, solubility, specific surface area, surface charge and surface reactivity constitute a more accurate measure of ENPs functions and toxic effects in addition to mass concentration. Effects of ENPs are also highly dependent on dose metrics, species and strains of organisms, environmental conditions, exposure route and duration. Searching correlations between properties and functions or biological effects may serve as an effective way in understanding positive and negative impacts of ENPs. This will ensure safe design and sustainable future use of ENPs.

Long QT syndrome caveolin-3 mutations differentially modulate Kv 4 and Cav 1.2 channels to contribute to action potential prolongation.

KEY POINTS: Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Cav 1.2-encoded L-type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv 4.2 and Kv 4.3 channels responsible for Ito . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating ICa,L but, for Cav3-S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches. ABSTRACT: Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Cav 1.2+Cav ß2cN4 channels, as well as Kv 4.2 and Kv 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca2+ -dependent inactivation of ICa,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+ -dependent inactivation of ICa,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

RATIONALE: Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. OBJECTIVE: We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. METHODS AND RESULTS: We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. CONCLUSIONS: Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

A Comparative Study on the Formation of Environmentally Persistent Free Radicals (EPFRs) on Hematite and Goethite: Contribution of Various Catechol Degradation Byproducts.

The formation and occurrence of environmentally persistent free radicals (EPFRs) have recently attracted increasing research attention. The interactions between organics and transition metals and the crystalline forms of the transition metals are essential for EPFR formation. This study is thus designed to investigate catechol degradation and compare the characteristics of EPFRs on α-Fe2O3 (hematite, HM) and α-FeOOH (goethite, GT). Catechol degradation was inhibited in the dark in the presence of iron oxides. The inhibition was stronger on GT-silica, but the electron paramagnetic resonance (EPR) signals of the two systems were comparable. The enhanced degradation under UV light irradiation was comparable between HM-silica and GT-silica, but the EPR signals were stronger on GT-silica. Catechol was adsorbed on HM in a mononuclear bidentate (M-B) configuration, but it was adsorbed in both mononuclear monodentate (M-M) and binuclear bidentate (B-B) configurations on GT. After series analysis, we proposed that the dimer-type radical (2,2',3,3'-tetrahydroxy-1,1'-biphenylene) was responsible for the more stable EPR signals for the HM system, while the M-M structure was more favorable for the catechol stabilization. Note that in the analysis of EPFR formation mechanisms, it is important to consider (1) different crystalline lattices and (2) the contribution of the degradation byproducts of the parent organics.

Homo-Conjugation of Low Molecular Weight Organic Acids Competes with Their Complexation with Cu(II).

Dissolved organic matter (DOM) controls the bioavailability and toxicity of heavy metals in aquatic environments. The observation of decreased conditional binding constants with increasing DOM concentration is not well documented, which may result in significant uncertainties in heavy metal behavior modeling and risk assessment. We used eight low molecular weight organic acids (LMOC) with representative structures to simulate DOM molecules. The interactions between LMOC molecules resulted in the decreased Cu(II)-LMOC binding with increasing LMOC concentrations, but higher pH values than theoretical calculation after mixing LMOC solutions of different pHs. We thus proposed homoconjugation between LMOC molecules through negative charge-assisted H-bond ((-)CAHB). A mathematic model was developed to describe Cu(II)-LMOC complexation ( KC) and LMOC homoconjugation ( KLHL). The increased competition of LMOC homoconjugation over Cu(II)-LMOC complexation, as suggested by the increased ratios of KLHL/ KC, resulted in the apparently decreased Cu(II)-LMOC binding with the increased LMOC concentration. Similar concentration-dependent binding was also observed for DOM. With the identified homoconjugation between DOM molecules, some of the literature data with concentration-dependent behavior may be re-evaluated. This is the first work that quantitatively identified homoconjugation among organic molecules. Both the modeling concepts and results provide useful information in investigating the environmental roles of DOM in mediating metal bioavailability and transport.

Junctional cleft [Ca²âº]i measurements using novel cleft-targeted Ca²âº sensors.

RATIONALE: Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum Ca(2+) release and signaling are controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional sarcoplasmic reticulum. However, methods to measure [Ca(2+)]Cleft directly are needed. OBJECTIVE: To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. METHODS AND RESULTS: We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors. The fluorescence pattern, affinity for ryanodine receptors, and competition by untagged FKBP12.6 demonstrated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic sarcoplasmic reticulum Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient with GCaMP2.2-FKBP12.6 versus GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L versus [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the sarcoplasmic reticulum Ca(2+) leak rather than sarcolemmal Ca(2+) flux. CONCLUSIONS: We have developed junctional cleft-targeted sensors to measure [Ca(2+)]Cleft versus [Ca(2+)]Bulk and demonstrated dynamic differences during electric excitation and a standing diastolic [Ca(2+)]i gradient, which could influence local Ca(2+)-dependent signaling within the junctional cleft.

Can the concentration of environmentally persistent free radicals describe its toxicity to Caenorhabditis elegans? Evidence provided by neurotoxicity and oxidative stress.

Environmentally persistent free radicals (EPFRs) are emerging pollutants stabilized on or inside particles. Although the toxicity of EPFR-containing particles has been confirmed, the conclusions are always ambiguous because of the presence of various compositions. A clear dose-response relationship was always challenged by the fact that the concentrations of these coexisted components simultaneously changed with EPFR concentrations. Without these solid dose-response pieces of evidence, we could not confidently conclude the toxicity of EPFRs and the description of potential EPFR risks. In this study, we established a particle system with a fixed catechol concentration but different reaction times to obtain particles with different EPFR concentrations. Caenorhabditis elegans (C. elegans) in response to different EPFR concentrations was systematically investigated at multiple biological levels, including behavior observations and biochemical and transcriptome analyses. Our results showed that exposure to EPFRs disrupted the development and locomotion of C. elegans. EPFRs cause concentration-dependent neurotoxicity and oxidative damage to C. elegans, which could be attributed to reactive oxygen species (ROS) promoted by EPFRs. Furthermore, the expression of key genes related to neurons was downregulated, whereas antioxidative genes were upregulated. Overall, our results confirmed the toxicity from EPFRs and EPFR concentration as a rational parameter to describe the extent of toxicity.

Modeling Idiopathic Ventricular Fibrillation Using iPSC Cardiomyocytes and Computational Approaches: A Proof-of-Concept Study.

Idiopathic ventricular fibrillation (IVF) is an unrefined diagnosis representing a heterogeneous patient group without a structural or genetic definition. IVF treatment is not mechanistic-based due to the lack of experimental patient-models. We sought to create a methodology to assess cellular arrhythmia mechanisms for IVF as a proof-of-concept study. Using IVF patient-specific induced pluripotent stem cell-derived cardiomyocytes, we integrate electrophysiological optical mapping with computational modeling to characterize the cellular phenotype. This approach flips the traditional paradigm using a biophysically detailed computational model to solve the problem inversely. Insight into the cellular mechanisms of this patient's IVF phenotype could also serve as a therapeutic testbed.

Caveolar Compartmentalization of Pacemaker Signaling is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

Background: Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Methods: Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. Results: In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (I f) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. Conclusions: SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Distribution of environmentally persistent free radicals in size-segregated PMs emitted from residential biomass fuel combustion.

Environmentally persistent free radicals (EPFRs) are considered as an emerging pollutant due to their potential environmental risks, but the distribution characteristics of particulate matters (PMs)-EPFRs from residential combustion source are poorly understood. In this study, biomass (corn straw, rice straw, pine and jujube wood) combustion was studied in lab-controlled experiments. More than 80% of PM-EPFRs were distributed in PMs with aerodynamic diameter (dae) ≤ 2.1 µm, and their concentration in fine PMs was about 10 times that in coarse PM (2.1 µm ≤ dae ≤ 10 µm). The detected EPFRs were carbon-centered free radicals adjacent to oxygen atoms or a mixture of oxygen- and carbon-centered radicals. The concentrations of EPFRs in coarse and fine PMs were positively correlated with char-EC, but the EPFRs in fine PMs exhibited a negative correlation with soot-EC (p < 0.05). The increase of PM-EPFRs signals with the increased dilution ratio during pine wood combustion was more significant than that from rice straw, which may be resulted from the interactions between the condensable volatiles and the transition metals. Our study provides useful information for better understanding the formation of combustion-derived PM-EPFRs, and will be instructive for its purposeful emissions control.

The dual effect of disodium anthraquinone-2,6-disulfonate (AQDS) on the Cr(VI) removal by biochar: The enhanced electron transfer and the inhibited adsorption.

Due to large specific surface area, abundant surface functional groups, and stable chemical structure, biochar has been widely used in many environmental fields, including the remediation of Cr pollution. Alternatively, electrochemically active organic matter (e-OM), which is prevalent in both natural environments and industrial wastewater, exerts an inevitable influence on the mechanisms underlying Cr(VI) removal by biochar. The synergistic interplay between biochar and e-OM in the context of Cr(VI) remediation remains to be fully elucidated. In this study, disodium anthraquinone-2,6-disulfonate (AQDS) was used as a model for e-OM, characterized by its quinone group's ability to either donate or accept electrons. We found that AQDS sped up the Cr(VI) removal process, but the enhancement effect decreased with the increase in pyrolysis temperature. With the addition of AQDS, the removal amount of Cr(VI) by BC300 and BC600 increased by 160.0% and 49.5%, respectively. AQDS could release more electrons trapped in the lower temperature biochar samples (BC300 and BC600) for Cr(VI) reduction. However, AQDS inhibited the Cr(VI) removal by BC900 due to the adsorption of AQDS on biochar surface. In the presence of the small molecule carbon source lactate, more AQDS was adsorbed onto the biochar surface. This led to an inhibition of the electron transfer between biochar and Cr(VI), resulting in an inhibitory effect. This study has elucidated the electron transfer mechanism involved in the removal of Cr(VI) by biochar, particularly in conjunction with e-OM. Furthermore, it would augment the efficacy of biochar in applications targeting the removal of heavy metals.

Cholecystokinin-A Signaling Regulates Automaticity of Pacemaker Cardiomyocytes.

Aims: The behavior of pacemaker cardiomyocytes (PCs) in the sinoatrial node (SAN) is modulated by neurohormonal and paracrine factors, many of which signal through G-protein coupled receptors (GPCRs). The aims of the present study are to catalog GPCRs that are differentially expressed in the mammalian SAN and to define the acute physiological consequences of activating the cholecystokinin-A signaling system in isolated PCs. Methods and Results: Using bulk and single cell RNA sequencing datasets, we identify a set of GPCRs that are differentially expressed between SAN and right atrial tissue, including several whose roles in PCs and in the SAN have not been thoroughly characterized. Focusing on one such GPCR, Cholecystokinin-A receptor (CCK A R), we demonstrate expression of Cckar mRNA specifically in mouse PCs, and further demonstrate that subsets of SAN fibroblasts and neurons within the cardiac intrinsic nervous system express cholecystokinin, the ligand for CCK A R. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCK A R, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCK A R. The effect of sCCK-8 on firing rate is mediated by reduction in the rate of spontaneous phase 4 depolarization of PCs and is mitigated by activation of beta-adrenergic signaling. Conclusions: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the the cholecystokinin-A signaling pathway regulates PC automaticity.