Release Events Research Articles

Loss of Ca2+ spark‐to‐BK channel coupling impairs cerebral autoregulation in hypertension

IntroductionA reduction in blood flow to the brain in hypertension leads to vascular dementia, but what drives this is unknown. In vascular smooth muscle cells (VSMCs) the close spatial coupling (~20 nm) between Ca2+ release events from the sarcoplasmic reticulum (SR) (Ca2+ sparks) and the vasodilatory large‐conductance Ca2+ activated K+ (BK) channels, buffers pressure induced constriction and ensures correct cerebral perfusion.HypothesisImpaired BK channel activation was responsible for the impaired cerebral autoregulation in hypertension‐induced dementia.MethodsMale hypertensive mice (BPH/2) and the normotensive control strain (BPN/3) were euthanized in adhered to the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Experiments were carried out using pressure myography, confocal microscopy and electrophysiology techniques.ResultsPressure‐induced contraction was studied at a range of intraluminal pressures (10‐120 mmHg), and cerebral arteries from BPH/2 mice showed a more contractile phenotype compared to BPN/3 controls, confirming impaired autoregulation. BK channel activation by Ca2+ sparks was recorded using perforated patch electrophysiology to record spontaneous transient outward currents (STOCs). STOCs recorded from VSMCs from BPH/2 cerebral arteries, showed a significant reduction in both frequency and amplitude. To determine the mechanism behind this loss of BK activity, we first studied Ca2+ sparks in pressurised cerebral arteries loaded with Fluro‐4‐AM and found no difference between BPH/2 and BPN/3 mice. Next, we looked at BK alpha subunit expression, by recording whole cell BK currents with a fixed [Ca2+] in the pipette solution. Paradoxically, there was an increase in BK current density in the VSMC from the BPH/2 mice compare to BPN/3 mice. We then looked at the Ca2+ sensitivity of the BK channel using single channel electrophysiology, but again there were no differences between the BPH/2 and BPN/3 groups. Subsequently we used live‐cell fluorescent membrane dyes to look at the coupling between the SR (ER‐Tracker) and the plasma membrane (CellMask) in freshly isolated VSMCs. 3D reconstruction of the deconvolved images showed a reduction in both the coupling site volume and frequency in cells isolated from BPH/2 mice.ConclusionsThis data suggests that an increased spatial distance between the SR and the plasma membrane reduces [Ca2+] around the BK channel resulting in increased constriction of the arteries. Opportunities to restore the spark‐to‐BK coupling could be a novel approach to prevent vascular dementia caused by hypertension.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

PO-645-01 SUBTHRESHOLD DELAYED AFTERDEPOLARIZATIONS MEDIATED BY REDUCED TISSUE COUPLING PROVIDE AN IMPORTANT SUBSTRATE FOR UNIDIRECTIONAL BLOCK AND ARRHYTHMOGENESIS IN THE INFARCT BORDER ZONE

Delayed afterdepolarizations (DADs) caused by spontaneous calcium release (SCR) events have been implicated in arrhythmia formation in the border zone (BZ) of infarcted hearts. DADs may inactivate sodium channels forming a substrate for unidirectional conduction block. The role played by infarct anatomy and altered intracellular coupling in facilitating this phenomenon is not well understood.

The effects of driving time scales on coronal heating in a stratified atmosphere

Aims. We investigate the atmospheric response to coronal heating driven by random velocity fields with different characteristic time scales and amplitudes. Methods. We conducted a series of three-dimensional magnetohydrodynamic simulations of random driving imposed on a gravitationally stratified model of the solar atmosphere. In order to understand differences between alternating current (AC) and direct current (DC) heating, we considered the effects of changing the characteristic time scales of the imposed velocities. We also investigated the effects of the magnitude of the velocity driving. Results. In all cases, complex foot point motions lead to a proliferation of current sheets and energy dissipation throughout the coronal volume. For a given driving amplitude, DC driving typically leads to a greater rate of energy injection when compared to AC driving. This ultimately leads to the formation of larger currents, increased heating rates, and higher coronal temperatures in DC simulations. There is no difference in the spatial distribution of energy dissipation across simulations; however, energy release events in AC cases tend to be more frequent and last for less time than in DC cases. This results in more asymmetric temperature profiles for field lines heated by AC drivers. Higher velocity driving is associated with larger currents, higher temperatures, and the corona occupying a larger fraction of the simulation volume. In all cases, the majority of heating is associated with small energy release events, which occur much more frequently than larger events. Conclusions. When combined with observational results that highlight a greater abundance of oscillatory power in lower frequency modes, these findings suggest that energy release in the corona is more likely to be driven by longer time scale motions. In the corona, AC and DC driving occur concurrently and their effects remain difficult to isolate. The distribution of field line temperatures and the asymmetry of temperature profiles may reveal the frequency and longevity of energy release events and therefore the relative importance of AC and DC heating.

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?

Extraintestinal pathogenic Escherichia coli utilizes the surface-expressed elongation factor Tu to bind and acquire iron from holo-transferrin

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is a common anthropozoonotic pathogen that causes systemic infections. To establish infection, ExPEC must utilize essential nutrients including iron from the host. Transferrin is an important iron source for multiple bacteria. However, the mechanism by which ExPEC utilizes transferrin remains unclear. In this study, we found that iron-saturated holo-transferrin rather than iron-free apo-transferrin promoted the vitality of ExPEC in heat-inactivated human serum. The multifunctional protein Elongation factor Tu (EFTu) worked as a holo-transferrin binding protein. EFTu not only bound holo-transferrin rather than apo-transferrin but also released transferrin-related iron, with all domains of EFTu involved in holo-transferrin binding and iron release events. We also identified the surface location of EFTu on ExPEC. Overexpression of EFTu on the surface of nonpathogenic E. coli not only promoted the binding of bacteria to holo-transferrin but also facilitated the uptake of transferrin-related iron. More importantly, it significantly enhanced the survival of E. coli in heat-inactivated human serum, which was positively correlated with holo-transferrin but not apo-transferrin. Our research revealed a novel function of EFTu in binding holo-transferrin to promote iron uptake by bacteria, suggesting that EFTu was a potential virulence factor of ExPEC. In addition, our study provided research avenues into the iron acquisition and pathogenicity mechanisms of ExPEC.

Gambierol Blocks a K+ Current Fraction without Affecting Catecholamine Release in Rat Fetal Adrenomedullary Cultured Chromaffin Cells.

Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19–F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of KV channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K+ current and slowed the kinetics of K+ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K+ (KCa) and ATP-sensitive K+ (KATP) channels. The gambierol concentration necessary to inhibit 50% of the K+ current-component sensitive to the polyether (IC50) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by KCa channels in the control of exocytosis from fetal (F19–F20) adrenomedullary chromaffin cells.

Episodic massive release of methane during the mid-Cretaceous greenhouse

Abstract Methane-derived carbonates (MDCs) are common along modern and ancient continental margins, and the majority of such formations are associated with seafloor cold seeps. Here, we document petrographic, rare earth element + yttrium (REE + Y), carbonate clumped isotope temperature (TΔ47), and carbon-isotopic evidence from a shale succession in southern Tibet spanning a ~28 m.y. interval (ca. 113–85 Ma) that coincided with the mid-Cretaceous greenhouse event. At least nine limestone nodule-bearing horizons exhibit seep-associated sedimentary structures, including carbonate fabrics (e.g., micritic crusts, crystal fans, and botryoidal textures) and 13C-depleted isotopic compositions (δ13Ccarb < –32.3‰), which are indicative of methane-derived carbon sources. Along with sedimentary evidence, the patterns of TΔ47–δ13Ccarb–δ18Ocarb support precipitation of these MDCs over a large temperature range. The REE + Y compositions and europium (Eu) anomalies indicate that the release of methane was associated with hydrothermal fluids. Methane may have been derived from both thermogenic and biogenic sources based on the inorganic carbon isotopic signatures of the carbonate. These nodular carbonate horizons document multiple episodes of seafloor methane release during the mid-Cretaceous and represent exceptionally long-lived, active methane seepage. Massive methane release events may have played a role in generating the greenhouse climate of the mid-Cretaceous.

Discordant Ca2+ release in cardiac myocytes: characterization and susceptibility to pharmacological RyR2 modulation.

Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.

Wintertime Brine Discharge at the Surface of a Cold Polar Glacier and the Unexpected Absence of Associated Seismicity

AbstractA subglacial groundwater system beneath Taylor Glacier, Antarctica, discharges hypersaline, iron‐rich brine episodically at the glacier surface to create Blood Falls. However, the triggering mechanism for these brine release events is not yet understood. Identifying which fracture processes are observed seismically can help us better characterize the hydrological system at Taylor Glacier, and more generally, provide us with a broader understanding of englacial hydrologic activity in cold glaciers. We document wintertime brine discharge using time‐lapse photography. Subfreezing air temperatures during the brine discharge indicate that surface melt‐induced hydrofracture is an unlikely trigger for brine release. Further, we analyze local seismic data to test a hypothesis that fracturing generates elevated surface wave energy preceding and/or coinciding with brine release events. Our results show no discernible elevated Rayleigh wave activity prior to or during Blood Falls brine release. Instead, we find a pattern of seismic events dominated by a seasonal signal, with more Rayleigh events occurring in the summer than the winter from the Blood Falls source area. We calculate that the volumetric opening of cracks that would generate Rayleigh waves at our detection limits are of similar size to myriad cracks in glacier ice, lake ice, and frozen sediment in the terminus area. We therefore propose that any fracturing coincident with brine release activity likely consists of a series of smaller opening events that are masked by other seismicity in the local environment.

Z16b, a natural compound from Ganoderma cochlear is a novel RyR2 stabilizer preventing catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, lethal ventricular arrhythmia triggered by catecholamines. Mutations in genes that encode cardiac ryanodine receptor (RyR2) and proteins that regulate RyR2 activity cause enhanced diastolic Ca2+ release (leak) through the RyR2 channels, resulting in CPVT. Current therapies for CPVT are limited. We found that Z16b, a meroterpenoid isolated from Ganoderma cochlear, inhibited Ca2+ spark frequency (CaSF) in R2474S/ + cardiomyocytes in a dose-dependent manner, with an IC50 of 3.2 μM. Z16b also dose-dependently suppressed abnormal post-pacing Ca2+ release events. Intraperitoneal injection (i.p.) of epinephrine and caffeine stimulated sustained ventricular tachycardia in all R2474S/+ mice, while pretreatment with Z16b (0.5 mg/kg, i.p.) prevented ventricular arrhythmia in 9 of 10 mice, and Z16b administration immediately after the onset of VT abolished sVT in 9 of 12 mice. Of translational significance, Z16b significantly inhibited CaSF and abnormal Ca2+ release events in human CPVT iPS-CMs. Mechanistically, Z16b interacts with RyR2, enhancing the "zipping" state of the N-terminal and central domains of RyR2. A molecular docking simulation and point mutation and pulldown assays identified Z16b forms hydrogen bonds with Arg626, His1670, and Gln2126 in RyR2 as a triangle shape that anchors the NTD and CD interaction and thus stabilizes RyR2 in a tight "zipping" conformation. Our findings support that Z16b is a novel RyR2 stabilizer that can prevent CPVT. It may also serve as a lead compound with a new scaffold for the design of safer and more efficient drugs for treating CPVT.

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

Background: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. Methods: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. Results: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm−1s−1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [−0.1; 0.03], p = 0.387). Conclusions: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

The Importance of the Temporary Clip Removal Phase on Exposure to Hypoxia: On-Line Measurement of Temporal Lobe Oxygen Levels During Surgery for Middle Cerebral Artery Aneurysms.

Most studies concerning intraoperative temporary arterial occlusion overlook the period between and after clip placement. To analyze the brain tissue oxygen tension through the process by which anterograde arterial blood flow is re-established after temporary clipping (TR). In this prospective observational study, patients who presented to surgery for middle cerebral artery aneurysms were continuously monitored with ICM+, to obtain temporal (downstream) PbtO2 levels while M1 segment temporary clips were applied and removed. PbtO2 changes were analyzed and compared with the clipping phase, and measures of exposure to hypoxia were defined and assessed during both phases and used in a model to test the impact of extending them. Eighty-six TRs (20 patients) were recorded. The mean acquired amount of time per clip release (CR) event was 336.7 seconds. Temporary clip removal produced specifically shaped, highly individual PbtO2 curves that correlated with their corresponding clipping phase events but developing slower and less consistently. The CR phase was responsible for greater cumulative exposure to hypoxia than the clip application phase through the first and second minutes of each. In our model, the duration of the TR phase was mostly responsible for the total exposure to hypoxia, and longer CR phases reduced the mean exposure to hypoxia. During the clip removal phase, the brain tissue is still exposed to oxygen levels that are significantly below the baseline, reverting through a singular, dynamic process. Therefore, it must be regarded by surgeons with the same degree of attention as its counterpart.

A new software for detection of calcium sparks in cardiac myocytes

Calcium sparks are the elementary events of calcium release in cardiac myocytes and other cells, such as skeletal muscle or neurons. Measurements of spark properties are integral for our understanding of how cardiac diseases and/or pharmacological treatment affect the heart. Given their small size and short duration, sparks are typically imaged using the line-scan mode of confocal or multi-photon microscope. This yields a large amount of data which typically need to be processed automatically to save time and minimize the risk of researcher bias and/or mistake.

False confessions predict a delay between release from incarceration and official exoneration.

Little empirical research has examined postconviction processes associated with the unique legal events of release from incarceration and official exoneration. Across various models, we tested the influence of risk factors associated with wrongful convictions (false confessions, faulty or misleading forensic evidence, inadequate legal defense, mistaken eyewitness identifications, official misconduct, and perjury) and relevant alternative factors (e.g., presence of DNA, false guilty pleas, and race) on the exoneration process, with a particular focus on the role of false confessions. We expected that all risk factors would be meaningfully associated with the duration between wrongful conviction and release but that false confessions would be associated with longer delays between release and exoneration and would remain a meaningful predictor of the delay even when accounting for alternative factors. Using data from documented exonerations of murder, attempted murder, and accessory to murder in the National Registry of Exonerations (N = 1,074), we examined the association of risk factors and alternative predictors with the time between exonerees' wrongful conviction and release from incarceration and the time between release from incarceration and official exoneration. Overall, five of the six risk factors predicted the time between wrongful conviction and release from incarceration, but of the risk factors, only false confessions predicted the time between release and exoneration (d = 0.28; 95% CI [0.13, 0.43]), even when we controlled for relevant alternative factors (d = 0.29; 95% CI [0.14, 0.43]). Exonerations that involve false confessions are associated with delays in the critical window between innocent people's release and official exoneration-a time during which these innocent people are precluded from accessing reintegration aids and may struggle to find housing and employment. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Estimation of the Thyroid Equivalent Doses to Residents in Areas Affected by the 2011 Fukushima Nuclear Disaster Due to Inhalation of 131I Based on Their Behavioral Data and the Latest Atmospheric Transport and Dispersion Model Simulation.

It has been challenging to obtain reliable estimates of thyroid equivalent doses (TEDs) to residents involved in the 11 March 2011 Fukushima Daiichi Nuclear Power Plant (FDNPP) accident because of the shortage of direct human measurements associated with 131I, the largest contributor to the dose of concern. The present study attempted to perform the estimation of the TEDs by inhalation of 131I to residents from Namie-town, one of the most radiologically-affected municipalities, by means of the latest atmospheric transport and dispersion model (ATDM) simulations with the Worldwide version of System for Prediction of Environmental Emergency Dose Information (WSPEEDI) ver. 2, coupled with personal behavioral data containing the history of the whereabouts of individuals shortly after the accident. We analyzed 1,637 residents who underwent direct measurements with whole-body counters several months after the accident and provided their personal behavioral data. We divided the subjects into two groups based on whether the distances between their locations and the FDNPP were >20 km as of 15:00 on 12 March in relation to the timepoint of the hydrogen explosion at the Unit 1 Reactor building. As a result, the 90th percentile TEDs of the 1,249 prompt evacuees and 388 late evacuees were 3.9 mSv (adult)-6.8 mSv (10-y-old) and 24.1 mSv (adult)-35.6 mSv (5-y-old), respectively, excluding 16 persons whose TEDs exceeded 50 mSv. The 90th percentile (median) TEDs to 1-y-old children (not included in the subjects) for the prompt and late evacuation groups were 8.1 (1.0) mSv and 36.3 (19.7) mSv, respectively. Additionally, this study provided the evidence to support the view that the explosive event at the Unit 1 Reactor building on the afternoon of 12 March 2011 could have caused the critical group among Namie-town's residents, whereas the largest release event on 15 March gave relatively small doses to the residents because their exposure took place mostly at sites that were distant from the FDNPP. However, the present dose estimation has potentially large uncertainty at the individual level; further validations are thus necessary.

Pathologically increased RyR2-Nav1.6 colocalization in db/db mouse model of diabetic cardiomyopathy

Diabetes affects more than 6% of the world population and can lead to diabetic cardiomyopathy (DCM), a condition characterized by left ventricular hypertrophy, heart failure (HF), and eventual sudden cardiac death. Linking structural remodeling with phenotype progression in DCM will enable early diagnosis and treatment, which is key for patient longevity. Previous studies have linked pathological increases in late sodium current, mediated by voltage-gated sodium channel subtype 1.6 (NaV1.6) localized near ryanodine receptor (RyR2) calcium release channels and sodium-calcium exchangers, to arrhythmogenic calcium release events in multiple cardiac disorders.

Magnesium Ions Moderate Calcium-Induced Calcium Release in Cardiac Calcium Release Sites by Binding to Ryanodine Receptor Activation and Inhibition Sites.

Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.

A two-subpopulation model that reflects heterogeneity of large dense core vesicles in exocytosis

ABSTRACT Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s−1, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s−1) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.

The Market Impact of New Auditing Standards in China

To improve audit reports' transparency and information content, China implemented new auditing standards in 2017. The standards, which include disclosing key audit matters, were applied to A+H share companies in 2017 and A-share companies in 2018. This paper uses A+H-share companies as the experimental group and A-share companies as the control group and applies the difference-in-differences method to test the market response to the new auditing standards. We find a statistically significant positive cumulative abnormal return (CAR) during the report release event window for the A+H share companies. The results indicate a greater investor reaction to the release of audit reports under the new standards implying that the audit reforms enhanced the information of the reports. Furthermore, the positive CAR indicates that the reports reassured investors that the audit did not reveal significant problems or that existing weaknesses were addressed. We also found a statistically insignificant difference in the volatility of stock prices during the event window for the experimental and control group.