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.

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.

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.

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.

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.

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.

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.

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 (CCKAR), 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 CCKAR. Using mouse models, we find that while baseline SAN function is not dramatically affected by loss of CCKAR, the firing rate of individual PCs is slowed by exposure to sulfated cholecystokinin-8 (sCCK-8), the high affinity ligand for CCKAR. 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. Conclusion: (1) PCs express many GPCRs whose specific roles in SAN function have not been characterized, (2) Activation of the cholecystokinin-A signaling pathway regulates PC automaticity.

A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene.

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.

Direct toxicity of environmentally persistent free radicals to nematode Caenorhabditis elegans after excluding the concomitant chemicals.

Environmentally persistent free radicals (EPFRs) have attracted extensive attention due to their potential toxicity. However, EPFRs-containing particles always coexist with their parent organic contaminants and intermediate degradation products (IM), which may have hindered the toxicity assessment of EPFRs. In this study, the toxicity of EFFRs was specifically verified after comparing the systems without EPFRs, such as the immediate mixture of catechol (CT) and particles, solutions of CT only, IM extracted from the particles, as well as particles after EPFRs quenching. Caenorhabditis elegans (C. elegans) were used as model organisms. Our results showed that EPFRs-containing particles (Si-Al-CT) exhibited significant toxicity to C. elegans, but not for the parent chemical CT and IM on the particles. Higher levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the Si-Al-CT system were attributed to the mediated generation of ·O2- and ·OH via EPFRs. EPFRs could increase gene expressions related not only to oxidative stress and biotransformation in C. elegans, but also to indications of disturbances in energy homeostasis, survival, proliferation, cell and embryonic development. Overall, these results confirmed the direct toxicity of EPFRs and highlighted the key role of EPFRs which may be neglected in assessing the environmental risks of organic contaminants.

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.

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.

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.

Persulfate adsorption and activation by carbon structure defects provided new insights into ofloxacin degradation by biochar.

Cellulose and lignin derived biochars with significant differences in persistent free radicals (PFRs), oxygen-containing functional groups, and defective structure were prepared to explore the mechanism of biochar mediated persulfate (PS) activation. EPR spin trapping and quenching technique coupled with degradation experiments confirmed that the defective structures could activate PS to generate superoxide anions (O2•-), which was converted to singlet oxygen (1O2), especially in the acidic condition. 1O2 dominated the degradation of ofloxacin (OFL, a fluoroquinolone antibiotic). An improved iodometric measurement was applied for direct quantification of adsorbed PS on biochar. The amounts of adsorbed PS were consistent with the degradation of OFL and the measured electric current during the reaction indicated that PS adsorption was a prerequisite for PS activation, which may be neglected in previous studies. The results of this study highlighted the key roles of defective structure and adsorption of PS on biochar for the activation of PS.

Competitive adsorption of methanol co-solvent and dioctyl phthalate on functionalized graphene sheet: Integrated investigation by molecular dynamics simulations and quantum chemical calculations.

HYPOTHESIS: Organic co-solvents, which are universally employed in adsorption studies of hydrophobic organic chemicals (HOCs), can inhibit HOC adsorption by competing for active sites on the adsorbent. The adsorbent structure can influence co-solvent interference of HOC adsorption; however, this effect remains unclear, leading to an incomplete understanding of the adsorption mechanism. EXPERIMENTS: In this study, dioctyl phthalate (DOP) was used to investigate competitive adsorption on functionalized graphene sheet in a water-methanol co-solvent system through molecular dynamics simulations and quantum chemical calculations. FINDINGS: The simulations showed that the functional groups in the graphene defects had a strong adsorption affinity for methanol. The adsorbed methanol occupied a large number of active sites at the graphene center, thereby weakening DOP adsorption. However, the methanol adsorbed at the graphene edges could not compete with DOP for the active sites. -COOH had the strongest binding affinity for methanol among the functional groups and thus predominantly controlled the interaction between graphene and methanol. This study makes an innovative contribution toward understanding the competitive adsorption of methanol and DOP on functionalized graphene sheet, especially in visualizing the competition for active sites, and provides theoretical guidance for the removal of HOCs and practical application of graphene.

Cellular and Molecular Mechanisms of Functional Hierarchy of Pacemaker Clusters in the Sinoatrial Node: New Insights into Sick Sinus Syndrome.

The sinoatrial node (SAN), the primary pacemaker of the heart, consists of a heterogeneous population of specialized cardiac myocytes that can spontaneously produce action potentials, generating the rhythm of the heart and coordinating heart contractions. Spontaneous beating can be observed from very early embryonic stage and under a series of genetic programing, the complex heterogeneous SAN cells are formed with specific biomarker proteins and generate robust automaticity. The SAN is capable to adjust its pacemaking rate in response to environmental and autonomic changes to regulate the heart's performance and maintain physiological needs of the body. Importantly, the origin of the action potential in the SAN is not static, but rather dynamically changes according to the prevailing conditions. Changes in the heart rate are associated with a shift of the leading pacemaker location within the SAN and accompanied by alterations in P wave morphology and PQ interval on ECG. Pacemaker shift occurs in response to different interventions: neurohormonal modulation, cardiac glycosides, pharmacological agents, mechanical stretch, a change in temperature, and a change in extracellular electrolyte concentrations. It was linked with the presence of distinct anatomically and functionally defined intranodal pacemaker clusters that are responsible for the generation of the heart rhythm at different rates. Recent studies indicate that on the cellular level, different pacemaker clusters rely on a complex interplay between the calcium (referred to local subsarcolemmal Ca2+ releases generated by the sarcoplasmic reticulum via ryanodine receptors) and voltage (referred to sarcolemmal electrogenic proteins) components of so-called "coupled clock pacemaker system" that is used to describe a complex mechanism of SAN pacemaking. In this review, we examine the structural, functional, and molecular evidence for hierarchical pacemaker clustering within the SAN. We also demonstrate the unique molecular signatures of intranodal pacemaker clusters, highlighting their importance for physiological rhythm regulation as well as their role in the development of SAN dysfunction, also known as sick sinus syndrome.

CuO and TiO2 particles generated more stable and stronger EPFRs in dark than under UV-irradiation.

Environmentally persistent free radicals (EFPRs) have recently attracted a great deal of research attention because of their significant toxicity and ubiquitous occurrence in the environment. The information is still very limited on how to estimate the intensity of EPFRs under ambient circumstances. This study is designed to specifically compare EPFRs generation during catechol degradation in dark and UV light irradiation. CuO and TiO2 were selected as model metal oxides to coat on silica at 1% CuO has a large electron exchange capacity, which may mediate catechol degradation in dark, while TiO2 possesses strong photocatalytic property and could accelerate catechol degradation under UV light. Under UV light irradiation, EPFRs were generated very quickly and reached the maximum value in 4 d, which was related to the photocatalytic property of the particle. However, these EPFRs dissipated quickly in 14 d. On the contrary, the intensities of EPFRs generated in dark were 2 times higher, and stabled for over 2 months. Therefore, the environmental impacts of EPFRs in dark may be widespread and long-lasting, which should be monitored more carefully. It should be noted that for CuO-coated silica, a significant amount of EPFRs (20% of the maximum) survived the UV-light irradiation and stabled during the experimental period (45 d). Stronger EPFRs were associated with more abundant dimer structures, suggesting the dimer structures were related to EPFRs formation during catechol degradation. Monitoring the generation of dimer structures in the degradation of organic chemicals may provide useful information to estimate EPFRs generation and risks.

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.