Spark Amplitude Research Articles

Measurements of sound propagation in Mars' lower atmosphere

Acoustics has become extraterrestrial and Mars provides a new natural laboratory for testing sound propagation models compared to those ones on Earth. Owing to the unique combination of a microphone and two sound sources, the Ingenuity helicopter and the SuperCam laser-induced sparks, the Mars 2020 Perseverance rover payload enables the in situ characterization of unique sound propagation properties of the low-pressure CO2-dominated Mars atmosphere. In this study, we show that atmospheric turbulence is responsible for a large variability in the sound amplitudes from laser-induced sparks. This variability follows the diurnal pattern of turbulence. In addition, acoustic measurements acquired over one Martian year reveal a variation of the sound intensity by a factor of 1.8 from a constant source due to the seasonal cycle of pressure and temperature that significantly modifies the acoustic impedance and shock-wave formation. Finally, we show that the evolution of the Ingenuity tones and laser spark amplitudes with distance is consistent with one of the existing sound absorption models, which is a key parameter for numerical simulations applied to geophysical experiments on CO2-rich atmospheres. Overall, these results demonstrate the potential of sound propagation to interrogate the Mars environment and will therefore help in the design of future acoustic-based experiments for Mars or other planetary atmospheres such as Venus and Titan.

Web Service Composition Optimization with the Improved Fireworks Algorithm

Even though the number of services is increasing, a single service can just complete simple tasks. In the face of complex tasks, we require composite multiple services to complete them. For the purpose of improving the efficiency of web service composition, we propose a service composition approach based on an improved fireworks algorithm (FWA++). First, we use the strategy of random selection to keep N − 1 individuals for the next generation, and the purpose is to speed up the convergence speed of the FWA++ and enhance the search ability. Second, we randomize the total number of sparks and maximum amplitude of sparks for each generation. In this way, the search ability and the ability to jump out of the local optimal solution are dynamically balanced throughout the execution of the algorithm. Our experimental results show that compared with other existing approaches, the approach proposed in this paper is more efficient and stable.

Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload.

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

Modulated Calcium Homeostasis and Release Events Under Atrial Fibrillation and Its Risk Factors: A Meta-Analysis

Background: Atrial fibrillation (AF) is associated with calcium (Ca2+) handling remodeling and increased spontaneous calcium release events (SCaEs). Nevertheless, its exact mechanism remains unclear, resulting in suboptimal primary and secondary preventative strategies.Methods: We searched the PubMed database for studies that investigated the relationship between SCaEs and AF and/or its risk factors. Meta-analysis was used to examine the Ca2+ mechanisms involved in the primary and secondary AF preventative groups.Results: We included a total of 74 studies, out of the identified 446 publications from inception (1982) until March 31, 2020. Forty-five were primary and 29 were secondary prevention studies for AF. The main Ca2+ release events, calcium transient (standardized mean difference (SMD) = 0.49; I2 = 35%; confidence interval (CI) = 0.33–0.66; p < 0.0001), and spark amplitude (SMD = 0.48; I2 = 0%; CI = −0.98–1.93; p = 0.054) were enhanced in the primary diseased group, while calcium transient frequency was increased in the secondary group. Calcium spark frequency was elevated in both the primary diseased and secondary AF groups. One of the key cardiac currents, the L-type calcium current (ICaL) was significantly downregulated in primary diseased (SMD = −1.07; I2 = 88%; CI = −1.94 to −0.20; p < 0.0001) and secondary AF groups (SMD = −1.28; I2 = 91%; CI = −2.04 to −0.52; p < 0.0001). Furthermore, the sodium–calcium exchanger (INCX) and NCX1 protein expression were significantly enhanced in the primary diseased group, while only NCX1 protein expression was shown to increase in the secondary AF studies. The phosphorylation of the ryanodine receptor at S2808 (pRyR-S2808) was significantly elevated in both the primary and secondary groups. It was increased in the primary diseased and proarrhythmic subgroups (SMD = 0.95; I2 = 64%; CI = 0.12–1.79; p = 0.074) and secondary AF group (SMD = 0.66; I2 = 63%; CI = 0.01–1.31; p < 0.0001). Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup.Conclusions: Our study identified that ICaL is reduced in both the primary and secondary diseased groups. Furthermore, pRyR-S2808 and NCX1 protein expression are enhanced. The remodeling leads to elevated Ca2+ functional activities, such as increased frequencies or amplitude of Ca2+ spark and Ca2+ transient. The main difference identified between the primary and secondary diseased groups is SERCA expression, which is elevated in the primary diseased group and substantially reduced in the secondary paroxysmal AF subgroup. We believe our study will add new evidence to AF mechanisms and treatment targets.

Alterations of Ca2+ signaling and Ca2+ release sites in cultured ventricular myocytes with intact internal Ca2+ storage

Although cultured adult cardiac myocytes in combination with cell-level genetic modifications have been adopted for the study of protein function, the cellular alterations caused by the culture conditions themselves need to be clarified before we can interpret the effects of genetically altered proteins. We systematically compared the cellular morphology, global Ca2+ signaling, elementary Ca2+ release (sparks), and arrangement of ryanodine receptor (RyR) clusters in short-term (2 days)-cultured adult rat ventricular myocytes with those of freshly isolated myocytes. The transverse (t)-tubules were remarkably decreased (to ∼25%) by culture, and whole-cell capacitance was reduced by ∼35%. The magnitude of depolarization-induced Ca2+ transients decreased to ∼50%, and Ca2+ transient decay was slowed by culture. The culture did not affect sarcoplasmic reticulum (SR) Ca2+ loading. Therefore, fractional Ca2+ release was attenuated by culture. In the cultured cells, the L-type Ca2+ current (ICa) was smaller (∼50% of controls) and its inactivation was slower. In cultured myocytes, there were significantly fewer (∼50% of control) Ca2+ sparks, the local Ca2+ releases through RyR clusters, compared with in freshly isolated cells. Amplitude and kinetics (duration and time-to-peak) of individual sparks were similar, but they showed greater width in cultured cells. Immunolocalization analysis revealed that the cross-striation of RyRs distribution became weaker and less organized, and that the density of RyR clusters decreased in cultured myocytes. Our data suggest that the loss of t-tubules and generation of compromised Ca2+ transients and ICa in short-term adult ventricular cell culture are independent of SR Ca2+ loading status. In addition, the deteriorated arrangement of the RyR-clusters and their decreased density after short-term culture may be partly responsible for fewer Ca2+ sparks and a decrease in global Ca2+ release.

IOCBIO Sparks detection and analysis software

Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development.

Iocbio Sparks Detection and Analysis Software

Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development.

High-intensity intermittent training is as effective as moderate continuous training, and not deleterious, in cardiomyocyte remodeling of hypertensive rats.

Exercise training offers possible nonpharmacological therapy for cardiovascular diseases including hypertension. High-intensity intermittent exercise (HIIE) training has been shown to have as much or even more beneficial cardiovascular effect in patients with cardiovascular diseases than moderate-intensity continuous exercise (CMIE) training. The aim of this study was to investigate the effects of the two types of training on cardiac remodeling of spontaneously hypertensive rats (SHR) induced by hypertension. Eight-week-old male SHR and normotensive Wistar-Kyoto rats (WKY) were divided into four groups: normotensive and hypertensive control (WKY and SHR-C) and hypertensive trained with CMIE (SHR-T CMIE) or HIIE (SHR-T HIIE). After 8 wk of training or inactivity, maximal running speed (MRS), arterial pressure, and heart weight were all assessed. CMIE or HIIE protocols not only increased final MRS and left ventricular weight/body weight ratio but also reduced mean arterial pressure compared with sedentary group. Then, left ventricular tissue was enzymatically dissociated, and isolated cardiomyocytes were used to highlight the changes induced by physical activity at morphological, mechanical, and molecular levels. Both types of training induced restoration of transverse tubule regularity, decrease in spark site density, and reduction in half-relaxation time of calcium transients. HIIE training, in particular, decreased spark amplitude and width, and increased cardiomyocyte contractility and the expression of sarco(endo)plasmic reticulum Ca2+-ATPase and phospholamban phosphorylated on serine 16. NEW & NOTEWORTHY High-intensity intermittent exercise training induces beneficial remodeling of the left ventricular cardiomyocytes of spontaneously hypertensive rats at the morphological, mechanical, and molecular levels. Results also confirm, at the cellular level, that this type of training, as it appears not to be deleterious, could be applied in rehabilitation of hypertensive patients.

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

BackgroundRyanodine receptors (RyR) mediate sarcoplasmic reticulum calcium (Ca2+) release and influence myocyte Ca2+ homeostasis and arrhythmias. In cardiac myocytes, RyRs are found in clusters of various sizes and shapes, and RyR cluster size may critically influence normal and arrhythmogenic Ca2+ spark and wave formation. However, the actual RyR cluster sizes at specific Ca2+ spark sites have never been measured in the physiological setting.Methods and ResultsHere we measured RyR cluster size and Ca2+ sparks simultaneously to assess how RyR cluster size influences Ca2+ sparks and sarcoplasmic reticulum Ca2+ leak. For small RyR cluster sizes (<50), Ca2+ spark frequency is very low but then increases dramatically at larger cluster sizes. In contrast, Ca2+ spark amplitude is nearly maximal even at relatively small RyR cluster size (≈10) and changes little at larger cluster size. These properties agreed with computational simulations of RyR gating within clusters.ConclusionsOur study explains how this combination of properties may limit arrhythmogenic Ca2+ sparks and wave propagation (at many junctions) while preserving the efficacy and spatial synchronization of Ca2+‐induced Ca2+‐release during normal excitation‐contraction coupling. However, variations in RyR cluster size among individual junctions and RyR sensitivity could exacerbate heterogeneity of local sarcoplasmic reticulum Ca2+ release and arrhythmogenesis under pathological conditions.

Performance evaluation methodology for multistage launch vehicles with high-fidelity modeling

Multistage launch vehicles of reduced size, such as ”Super Strypi” or ”Sword”, are currently investigated for the purpose of providing launch opportunities for microsatellites. Currently, microsatellites are launched according to timing and orbit requirements of the main payload. The limited costs of microsatellites and their capability to be produced and ready for use in short time make them particularly suitable for ready-on-demand requests, such as facing an emergency. As a result, launch vehicles for the exclusive use of microsatellites would be very useful. This work considers the Scout rocket, a four-stage launch vehicle of reduced size used in the past. Its aerodynamics and propulsion are modeled with high fidelity, through interpolation of reliable, accurate available data. For the purpose of reducing the rocket complexity and size, as well as the launch cost per kg of payload, simplification of the rocket subsystems is advisable, and this includes also the guidance system and the related algorithm. In fact, open-loop guidance was actually employed during real Scout flights. In this research, open-loop guidance is investigated, under the assumption that the aerodynamic angle of attack is constant for each of the first three stages. Instead, for the upper stage the terminal optimal ascent path leading to orbit injection is determined through the use of a specific implementation of firework algorithm, in conjunction with the Euler-Lagrange equations and the Pontryagin minimum principle. Firework algorithms represent a recently-introduced heuristic technique inspired by the firework explosions in the night sky. The concept that underlies this method is relatively simple: a firework explodes in the search space of the unknown parameters, with amplitude and number of sparks determined dynamically. The succeeding iterations preserve the best sparks. The firework algorithm has several original features that can ensure satisfactory performance in parameter optimization problems, because both local search and global search are effectively performed through combination of various stochastic operators. With regard to the problem at hand, the unknown parameters are (i) the aerodynamic angles of attack of the first three stages, (ii) the coast time interval and (iii) the initial values of the adjoint variables conjugate to the upper stage dynamics. The numerical results unequivocally prove that the methodology at hand is rather robust, effective, and accurate, and definitely allows evaluating the performance attainable from multistage launch vehicles with accurate aerodynamic and propulsive modeling.

Acute Hypoxia Alters Ryanodine Receptor Activity in Pulmonary Arterial Myocytes of High Altitude Acclimatized Fetal and Adult Sheep

Intrauterine stress, such as long term high altitude hypoxia (LTH), can alter pulmonary function and reprogram fetal pulmonary vascular development, potentially causing respiratory distress and pulmonary hypertension after birth. Our recent data demonstrates that newborn LTH lambs exhibit exaggerated hypoxia‐induced pulmonary vasoconstriction (HPV). Previous evidence shows Ryanodine Receptor (RyR) activation is central to the HPV response. Furthermore, LTH disrupts RyR activated Ca2+ sparks in pulmonary arterial (PA) myocytes of sheep. Given the relationship between RyR and HPV responses, we tested the hypothesis that acute hypoxia (exposure to 4% O2) and LTH increase RyR mediated Ca2+ spark activity. This was tested by measuring the Ca2+ spark activity in PA myocytes of near‐term fetal lambs and adult sheep that resided at either low (335m, LA) or high (3801m, HA) altitude. Intracellular Ca2+ was recorded in myocytes of isolated PA tissues loaded with Fluo‐4 using line‐scan techniques on a confocal microscope. Line‐scan images were analyzed using custom software (SparkLab) to determine the number of cells displaying sparks, spark frequency, and spatial‐ temporal characteristics. Adult myocytes had greater prevalence and frequency of Ca2+ sparks relative to those of fetuses, confirming previous studies. Acute hypoxia significantly decreased spark prevalence and frequency in myocytes from both HA fetal and LA adult groups; however, acute hypoxia increased spark prevalence and frequency in HA adult sheep. Spark amplitude was decreased by acute hypoxia in the LA fetus but increased in the HA adult. Acute hypoxia also reduced spark duration in the fetus independent of altitude but increased the duration in HA adult. These results provide new insight into the influence of high altitude reprogramming of RyR function in PA of the fetus and adult and the roles their activity have during acute hypoxic exposures.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. JT was a Loma Linda University Summer Undergraduate Research Fellow while TY was a Walter E Macpherson Society Medical Student Summer Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pregnancy Enhances Calcium Spark Activity Independent of Altitude in Ovine Uterine Arterial Myocytes

During pregnancy, uterine artery blood flow increases in order to accommodate the growing fetus. However, the molecular mechanisms by which this change in uterine blood flow occurs remains to be elucidated. The BKCa channel, which is activated in response to ryanodine receptor (RyR) generated increases in intracellular Ca2+ known as Ca2+ sparks, helps regulate uterine artery blood flow. BKCa channel activation leads to vascular smooth muscle relaxation, thus decreasing uterine artery vascular tone and increasing uterine blood flow during pregnancy. Further to this, long term hypoxia due to high altitude exposure diminishes pregnancy – related increases in uterine blood flow. Based on these relationships we hypothesized that both pregnancy and membrane depolarization would increase Ca2+ spark activity, whereas ryanodine and long term hypoxia would inhibit Ca2+ spark activity. Uterine arteries were isolated from near‐term pregnant (n=6) and non‐pregnant sheep (n=6) in normoxia, as well as near‐term pregnant (n=6) and non‐pregnant sheep (n=6) exposed to hypoxic conditions at White Mountain (elevation 3,801 m) for 100 plus days. Isolated uterine arteries were subjected to three distinct ex vivo treatments: (1) control (saline), (2) 30mM potassium (30K), which causes membrane depolarization, and (3) 30K with ryanodine (10 μM), an RyR antagonist. Indeed, Ca2+ spark activity was increased in pregnant uterine arteries as compared to non‐pregnant uterine arteries, whereas hypoxia reduced spark frequency in all the aforementioned groups. Treatment with 30K increased Ca2+ spark activity in all groups. In comparison, 30K + ryanodine treatment decreased Ca2+ spark activity. Pregnancy increased the full width of sparks. However, pregnancy or hypoxia caused only minor changes in the amplitude and temporal aspects of sparks. These findings support the claim that membrane depolarization, due to 30K treatment, causes an increase in Ca2+ spark activity whereas ryanodine inhibits release of Ca2+ from the sarcoplasmic reticulum and thus decreases Ca2+ sparks. Taken together, these findings support the notion that the ryanodine receptors and BKCa channels work in conjunction with each other. This study provides further insight into the mechanisms that regulate uterine artery vascular tone and blood flow, and thus may prove useful in designing treatments for pregnancy complications that arise from increased uterine vascular tone, such as intrauterine growth restriction.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. VW was a Walter E Macpherson Medical Student Summer Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study on volt-ampere characteristics of spark discharge for transistor resistor pulse power of EDM

Electrical discharge machining (EDM) is widely used in aerospace industry and difficult-to-machine materials field. But the research on machining mechanism is insufficient and it has become a bottleneck of restricting its further development. In this paper, spark discharge self-sustaining conditions, the effects of pulse power circuit parameters on gap volt-ampere characteristics of EDM, formation of maintaining voltage of spark discharge, frequency and amplitude fluctuation of high-frequency components of maintaining voltage, and stability of spark discharge state are studied. Forming process of maintaining voltage of spark discharge is analyzed through discharge main circuit structure of transistor resistor pulse power. Influence of current-limiting resistor on maintaining voltage of spark discharge is also studied. The effect law of current-limiting resistor on oscillation frequency of maintaining voltage of spark discharge is obtained through spectrum analysis of maintaining voltage of spark discharge by methods of fast Fourier transform (FFT). Gap spark discharge control system is established through discharge circuit model of transistor resistor pulse power and the gap of EDM is replaced by spark discharge gap equivalent form. Input of the control system is the voltage of pulse power and its output is the gap voltage. Influence of current-limiting resistor on stability and response speed of the system is analyzed. The above research is important for revealing the mechanism of EDM, maintaining the stability of spark discharge, and guiding the design of pulse power and selection of processing parameters.

Role of FK506-binding protein in Ca2+ spark regulation

The elementary Ca2+ release events, Ca2+ sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor (RyR) on the sarcoplasmic reticulum, remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein (FKBP), the role of FKBPs in modifying RyR Ca2+ sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca2+ sparks. In the present study, we detected Ca2+ sparks triggered by single L-type Ca2+ channels (LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca2+ spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca2+ in the sarcoplasmic reticulum, nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca2+ spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca2+ spark would be compromised despite the sensitization of individual RyRs.

Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies.

AimsIt is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests).Methods and resultsCa2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF.Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF.ConclusionCa2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses.

Mg2+ Inhibits Cardiac SR Calcium Release and has Biphasic Effects on Calmodulin Binding to RyR2

Mg2+ is a potent inhibitor of RyR channel gating, as demonstrated in lipid bilayer and SR vesicles. Less is known about Mg2+ effects on RyR2 function in the cardiac myocyte environment or how it might alter RyR2 interaction with accessory proteins like calmodulin (CaM), which by itself binds to, and inhibits RyR2 opening. We used permeabilized mouse ventricular cardiomyocytes to study the effects of Mg2+ on spontaneous Ca2+ release and CaM affinity for RyR2. We recorded Ca sparks at three [Mg2+]: 0.1 mM (low), 1 mM (physiological), and 3 mM (high). We found that in the presence of endogenous WT-CaM, with increasing [Mg2+], Ca spark frequency (CaSpF) decreased monotonically, and was accompanied by an increase in Ca spark amplitude and SR Ca2+ load. To test whether Mg2+ also modulates RyR2 interaction with accessory proteins, we measured binding affinity of WT-CaM and two CPVT-linked CaM mutants (N54I and N98S). The apparent affinity of WT-CaM for RyR2 in myocytes was highest at 1 mM Mg2+ and decreased at both 0.1 mM and 3 mM Mg2+. The same pattern was found for N54I- and N98S-CaM. We conclude that in cardiomyocytes, Mg2+ decreases CaSpF and increases SR Ca2+ load. As [Mg2+] increases in the physiologically range (0.1 - 1 mM), part of the Ca spark inhibition may be due to enhanced CaM binding to RyR2. In contrast, pathophysiologically high [Mg2+] (3 mM) may (1) limit RyR2-CaM binding (and thus CaM inhibition of RyR2) and (2) still directly inhibit RyR2 opening.

Modeling Na+-Ca2 + exchange in the heart: Allosteric activation, spatial localization, sparks and excitation-contraction coupling

The cardiac sodium (Na+)/calcium (Ca2+) exchanger (NCX1) is an electrogenic membrane transporter that regulates Ca2+ homeostasis in cardiomyocytes, serving mainly to extrude Ca2+ during diastole. The direction of Ca2+ transport reverses at membrane potentials near that of the action potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of NCX1 in cardiac Ca2+-induced Ca2+ release (CICR). Interest has been reinvigorated by a recent super-resolution optical imaging study suggesting that ~18% of NCX1 co-localize with ryanodine receptor (RyR2) clusters, and ~30% of additional NCX1 are localized to within ~120nm of the nearest RyR2. NCX1 may therefore occupy a privileged position in which to modulate CICR. To examine this question, we have developed a mechanistic biophysically-detailed model of NCX1 that describes both NCX1 transport kinetics and Ca2+-dependent allosteric regulation. This NCX1 model was incorporated into a previously developed super-resolution model of the Ca2+ spark as well as a computational model of the cardiac ventricular myocyte that includes a detailed description of CICR with stochastic gating of L-type Ca2+ channels and RyR2s, and that accounts for local Ca2+ gradients near the dyad via inclusion of a peri-dyadic (PD) compartment. Both models predict that increasing the fraction of NCX1 in the dyad and PD decreases spark frequency, fidelity, and diastolic Ca2+ levels. Spark amplitude and duration are less sensitive to NCX1 spatial redistribution. On the other hand, NCX1 plays an important role in promoting Ca2+ entry into the dyad, and hence contributing to the trigger for RyR2 release at depolarized membrane potentials and in the presence of elevated local Na+ concentration. Whole-cell simulation of NCX1 tail currents are consistent with the finding that a relatively high fraction of NCX1 (~45%) resides in the dyadic and PD spaces, with a dyad-to-PD ratio of roughly 1:2. Allosteric Ca2+ activation of NCX1 helps to “functionally localize” exchanger activity to the dyad and PD by reducing exchanger activity in the cytosol thereby protecting the cell from excessive loss of Ca2+ during diastole.

The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development.

Upon activation, mammalian eggs release billions of zinc ions in an exocytotic event termed the “zinc spark.” The zinc spark is dependent on and occurs coordinately with intracellular calcium transients, which are tightly associated with embryonic development. Thus, we hypothesized that the zinc spark represents an early extracellular physicochemical marker of the developmental potential of the zygote. To test this hypothesis, we monitored zinc exocytosis in individual mouse eggs following parthenogenetic activation or in vitro fertilization (IVF) and tracked their development. Retrospective analysis of zinc spark profiles revealed that parthenotes and zygotes that developed into blastocysts released more zinc than those that failed to develop. Prospective selection of embryos based on their zinc spark profile significantly improved developmental outcomes and more than doubled the percentage of embryos that reached the blastocyst stage. Moreover, the zinc spark profile was also associated with embryo quality as the total cell number in the resulting morulae and blastocysts positively correlated with the zinc spark amplitude (R = 0.9209). Zinc sparks can thus serve as an early biomarker of zygote quality in mouse model.

Simvastatin has Profound Effects on Sarcoplasmic Reticulum Ca2+ Leak in Skeletal but not Cardiac Muscle: A Mechanism for Myopathy

The primary reason for cessation of statin therapy is skeletal myopathy but the underlying mechanism and apparent absence of detrimental effects of statins on cardiac muscle are not fully understood. We have reported that statin treatment in vivo causes a profound leak of Ca2+ from the sarcoplasmic reticulum (SR) of intact skeletal myocytes, a common myopathic mechanism. Here we compare the effect of simvastatin on Ca2+ sparks in rat skeletal and cardiac myocytes. Simvastatin treatment in vivo (40 mg/kg/day for 4 weeks) markedly increased Ca2+ spark frequency (280%) and duration (140%) in fluo4-loaded flexor digitorum brevis (FDB)fibres (P<0.001), but had no effect on spark frequency and only a minor effect on duration (+26%; P<0.001) in ventricular myocytes. Spark amplitude and width were not changed in either cell type. Thus the profound statin-induced SR Ca2+ leak in skeletal muscle is absent in cardiac myocytes. To determine whether simvastatin acutely induces SR leak in skeletal muscle, saponin-permeabilised cells from control animals were perfused with 10 µM simvastatin carboxylate (the active form) for 5 min. In FDB, simvastatin increased spark frequency (38%; P<0.05) without significant effects on duration, amplitude or width. By contrast, in cardiac cells a minor reduction in frequency (17%) and amplitude (9%) were observed (P<0.01). Together, these data show that simvastatin has disparate effects on SR Ca2+ release in skeletal and cardiac muscle. A possible mechanism which contributes to statin effects on the skeletal myocyte is direct interaction of the drug with RyR. In support of this, simvastatin carboxylate (10 µM) increased the open probability of single RyR channels derived from skeletal, but not cardiac, muscle (see Venturi et al. this meeting).Funded by the BHF

Modeling the Role of NCX in the Regulation of Cardiomyocyte Microdomain Ca2+ Dynamics

The sodium (Na+)/calcium (Ca2+) exchanger (NCX) is an electrogenic membrane transporter that mainly serves as a Ca2+ removal mechanism during relaxation and maintains Ca2+ homeostasis in cardiomyocytes. The direction of transport reverses at membrane potentials near action the potential plateau, generating an influx of Ca2+ into the cell. Therefore, there has been great interest in the role NCX plays in cardiac Ca2+-induced Ca2+-release (CICR), with some controversial findings questioning whether NCX has any role at all. A recent super-resolution optical imaging study suggested that ∼17% of NCX co-localize within the domain occupied by ryanodine receptor (RyR) clusters and ∼30% of NCX are within 100nm of the dyad boundary. Therefore, NCX may play a critical role in modulating Ca2+ spark activity and CICR as a result of its spatial distribution in the T-tubule. Here we examine the potential role of NCX using computational modeling. We have developed a novel mechanistic and biophysically-detailed model of NCX that describes both NCX transport kinetics and dynamics of Ca2+-dependent allosteric regulation. We incorporated this new NCX model into a previously developed super-resolution Ca2+ spark model to examine effects of local NCX spatial distribution on properties of Ca2+ sparks. Dyadic NCX reduces spark frequency but has minimal effects on spark amplitude and half-width. Furthermore, we incorporate NCX into a previously developed computational model of the cardiac ventricular myocyte that includes a detailed description of CICR simulated with stochastic gating of L-Type Ca2+ channels and RyRs in the dyad and accounts for local Ca2+ gradients via inclusion of peri-dyadic compartments. We use this model to investigate the role of NCX and its localization on whole-cell Ca2+ dynamics, the Ca2+ levels in the junctional sarcoplasmic reticulum, and APs.