Local Control Model of a Human Ventricular Myocyte: An Exploration of Frequency-Dependent Changes and Calcium Sparks.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted "nonconducting" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.

Structural Analysis of Mitochondria in Cardiomyocytes: Insights into Bioenergetics and Membrane Remodeling.

Mitochondria in mammalian cardiomyocytes display considerable structural heterogeneity, the significance of which is not currently understood. We use electron microscopic tomography to analyze a dataset of 68 mitochondrial subvolumes to look for correlations among mitochondrial size and shape, crista morphology and membrane density, and organelle location within rat cardiac myocytes. A tomographic analysis guided the definition of four classes of crista morphology: lamellar, tubular, mixed and transitional, the last associated with remodeling between lamellar and tubular cristae. Correlations include an apparent bias for mitochondria with lamellar cristae to be located in the regions between myofibrils and a two-fold larger crista membrane density in mitochondria with lamellar cristae relative to mitochondria with tubular cristae. The examination of individual cristae inside mitochondria reveals local variations in crista topology, such as extent of branching, alignment of fenestrations and progressive changes in membrane morphology and packing density. The findings suggest both a rationale for the interfibrillar location of lamellar mitochondria and a pathway for crista remodeling from lamellar to tubular morphology.

Understanding the Dynamics of the Transient and Permanent Opening Events of the Mitochondrial Permeability Transition Pore with a Novel Stochastic Model.

The mitochondrial permeability transition pore (mPTP) is a non-selective pore in the inner mitochondrial membrane (IMM) which causes depolarization when it opens under conditions of oxidative stress and high concentrations of Ca2+. In this study, a stochastic computational model was developed to better understand the dynamics of mPTP opening and closing associated with elevated reactive oxygen species (ROS) in cardiomyocytes. The data modeled are from "photon stress" experiments in which the fluorescent dye TMRM (tetramethylrhodamine methyl ester) is both the source of ROS (induced by laser light) and sensor of the electrical potential difference across the IMM. Monte Carlo methods were applied to describe opening and closing of the pore along with the Hill Equation to account for the effect of ROS levels on the transition probabilities. The amplitude distribution of transient mPTP opening events, the number of transient mPTP opening events per minute in a cell, the time it takes for recovery after transient depolarizations in the mitochondria, and the change in TMRM fluorescence during the transition from transient to permanent mPTP opening events were analyzed. The model suggests that mPTP transient open times have an exponential distribution that are reflected in TMRM fluorescence. A second multiple pore model in which individual channels have no permanent open state suggests that 5-10 mPTP per mitochondria would be needed for sustained mitochondrial depolarization at elevated ROS with at least 1 mPTP in the transient open state.

Optimizing peptide inhibitors of SARS-Cov-2 nsp10/nsp16 methyltransferase predicted through molecular simulation and machine learning.

Coronaviruses, including the recent pandemic strain SARS-Cov-2, use a multifunctional 2'-O-methyltransferase (2'-O-MTase) to restrict the host defense mechanism and to methylate RNA. The nonstructural protein 16 2'-O-MTase (nsp16) becomes active when nonstructural protein 10 (nsp10) and nsp16 interact. Novel peptide drugs have shown promise in the treatment of numerous diseases and new research has established that nsp10 derived peptides can disrupt viral methyltransferase activity via interaction of nsp16. This study had the goal of optimizing new analogous nsp10 peptides that have the ability to bind nsp16 with equal to or higher affinity than those naturally occurring. The following research demonstrates that in silico molecular simulations can shed light on peptide structures and predict the potential of new peptides to interrupt methyltransferase activity via the nsp10/nsp16 interface. The simulations suggest that misalignments at residues F68, H80, I81, D94, and Y96 or rotation at H80 abrogate MTase function. We develop a new set of peptides based on conserved regions of the nsp10 protein in the Coronaviridae species and test these to known MTase variant values. This results in the prediction that the H80R variant is a solid new candidate for potential new testing. We envision that this new lead is the beginning of a reputable foundation of a new computational method that combats coronaviruses and that is beneficial for new peptide drug development.

Active site prediction of phosphorylated SARS-CoV-2 N-Protein using molecular simulation.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-protein) is responsible for viral replication by assisting in viral RNA synthesis and attaching the viral genome to the replicase-transcriptase complex (RTC). Numerous studies suggested the N-protein as a drug target. However, the specific N-protein active sites for SARS-CoV-2 drug treatments are yet to be discovered. The purpose of this study was to determine active sites of the SARS-CoV-2 N-protein by identifying torsion angle classifiers for N-protein structural changes that correlated with the respective angle differences between the active and inactive N-protein. In the study, classifiers with a minimum accuracy of 80% determined from molecular simulation data were analyzed by Principal Component Analysis and cross-validated by Logistic Regression, Support Vector Machine, and Random Forest Classification. The ability of torsion angles ψ252 and φ375 to differentiate between phosphorylated and unphosphorylated structures suggested that residues 252 and 375 in the RNA binding domain might be important in N-protein activation. Furthermore, the φ and ψ angles of residue S189 correlated to a 90.7% structural determination accuracy. The key residues involved in the structural changes identified here might suggest possible important functional sites on the N-protein that could be the focus of further study to understand their potential as drug targets.

Understanding Calmodulin Variants Affecting Calcium-Dependent Inactivation of L-Type Calcium Channels through Whole-Cell Simulation of the Cardiac Ventricular Myocyte.

Mutations in the calcium-sensing protein calmodulin (CaM) have been linked to two cardiac arrhythmia diseases, Long QT Syndrome 14 (LQT14) and Catecholaminergic Polymorphic Ventricular Tachycardia Type 4 (CPVT4), with varying degrees of severity. Functional characterization of the CaM mutants most strongly associated with LQT14 show a clear disruption of the calcium-dependent inactivation (CDI) of the L-Type calcium channel (LCC). CPVT4 mutants on the other hand are associated with changes in their affinity to the ryanodine receptor. In clinical studies, some variants have been associated with both CPVT4 and LQT15. This study uses simulations in a model for excitation-contraction coupling in the rat ventricular myocytes to understand how LQT14 variant might give the functional phenotype similar to CPVT4. Changing the CaM-dependent transition rate by a factor of 0.75 corresponding to the D96V variant and by a factor of 0.90 corresponding to the F142L or N98S variants, in a physiologically based stochastic model of the LCC prolonger, the action potential duration changed by a small amount in a cardiac myocyte but did not disrupt CICR at 1, 2, and 4 Hz. Under beta-adrenergic simulation abnormal excitation-contraction coupling was observed above 2 Hz pacing for the mutant CaM. The same conditions applied under beta-adrenergic stimulation led to the rapid onset of arrhythmia in the mutant CaM simulations. Simulations with the LQT14 mutations under the conditions of rapid pacing with beta-adrenergic stimulation drives the cardiac myocyte toward an arrhythmic state known as Ca2+ overload. These simulations provide a mechanistic link to a disease state for LQT14-associated mutations in CaM to yield a CPVT4 phenotype. The results show that small changes to the CaM-regulated inactivation of LCC promote arrhythmia and underscore the significance of CDI in proper heart function.

Machine learning-based prediction of drug and ligand binding in BCL-2 variants through molecular dynamics.

Venetoclax is a BH3 (BCL-2 Homology 3) mimetic used to treat leukemia and lymphoma by inhibiting the anti-apoptotic BCL-2 protein thereby promoting apoptosis of cancerous cells. Acquired resistance to Venetoclax via specific variants in BCL-2 is a major problem for the successful treatment of cancer patients. Replica exchange molecular dynamics (REMD) simulations combined with machine learning were used to define the average structure of variants in aqueous solution to predict changes in drug and ligand binding in BCL-2 variants. The variant structures all show shifts in residue positions that occlude the binding groove, and these are the primary contributors to drug resistance. Correspondingly, we established a method that can predict the severity of a variant as measured by the inhibitory constant (Ki) of Venetoclax by measuring the structure deviations to the binding cleft. In addition, we also applied machine learning to the phi and psi angles of the amino acid backbone to the ensemble of conformations that demonstrated a generalizable method for drug resistant predictions of BCL-2 proteins that elucidates changes where detailed understanding of the structure-function relationship is less clear.

Effect of crista morphology on mitochondrial ATP output: A computational study.

Folding of the mitochondrial inner membrane (IM) into cristae greatly increases the ATP-generating surface area, S IM, per unit volume but also creates diffusional bottlenecks that could limit reaction rates inside mitochondria. This study explores possible effects of inner membrane folding on mitochondrial ATP output, using a mathematical model for energy metabolism developed by the Jafri group and two- and three-dimensional spatial models for mitochondria, implemented on the Virtual Cell platform. Simulations demonstrate that cristae are micro-compartments functionally distinct from the cytosol. At physiological steady states, standing gradients of ADP form inside cristae that depend on the size and shape of the compartments, and reduce local flux (rate per unit area) of the adenine nucleotide translocase. This causes matrix ADP levels to drop, which in turn reduces the flux of ATP synthase. The adverse effects of membrane folding on reaction fluxes increase with crista length and are greater for lamellar than tubular crista. However, total ATP output per mitochondrion is the product of flux of ATP synthase and S IM which can be two-fold greater for mitochondria with lamellar than tubular cristae, resulting in greater ATP output for the former. The simulations also demonstrate the crucial role played by intracristal kinases (adenylate kinase, creatine kinase) in maintaining the energy advantage of IM folding.

Predicting Genetic Variation Severity Using Machine Learning to Interpret Molecular Simulations.

Distinct missense mutations in a specific gene have been associated with different diseases as well as differing severity of a disease. Current computational methods predict the potential pathogenicity of a missense variant but fail to differentiate between separate disease or severity phenotypes. We have developed a method to overcome this limitation by applying machine learning to features extracted from molecular dynamics simulations, creating a way to predict the effect of novel genetic variants in causing a disease, drug resistance, or another specific trait. As an example, we have applied this novel approach to variants in calmodulin associated with two distinct arrhythmias as well as two different neurodegenerative diseases caused by variants in amyloid-ß peptide. The new method successfully predicts the specific disease caused by a gene variant and ranks its severity with more accuracy than existing methods. We call this method molecular dynamics phenotype prediction model.

T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function.

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the store, store-operated Ca2+ entry (SOCE), and mitochondrial inner membrane potential (ΔΨm) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca2+]i and ΔΨm dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca2+]i, diminished responses to Ca2+ mobilization with thapsigargin, and decreased rate of [Ca2+]i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca2+]i and ability of thapsigargin to mobilize Ca2+ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨm in WT T cells, it produced ΔΨm hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca2+ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca2+ level to the amplitude of thapsigargin-induced Ca2+ transient and an integral of changes in ΔΨm in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca2+ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca2+ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.

Neuropeptide S receptor gene Asn107 polymorphism in obese male individuals in Pakistan.

Neuropeptide S (NPS) is a naturally occurring appetite stimulant, associated with anxiety, stress, and excitement regulation. Neuropeptide S serves as a hypothalamic energy regulator that enhances food intake with a reduced level of satiety. NPS activates fat angiogenesis and the proliferation of new adipocytes in obesity. NPS has an established role in energy regulation by many pre-clinical investigations; however we have limited data available to support this notion in humans. We found significant association of Neuropeptide S receptor (NPSR1) Asn107Ile (rs324981, A>T) polymorphism with obese male participants. The current investigation carried out genotype screening of NPSR1 allele to assess the spectrum of the Asn107Ile polymorphism in obese and healthy Pakistani individuals. We revealed a significant (p = 0.04) difference between AA vs TT + AT genotype distribution of NPSR1 (SNP rs324981,) between obese and healthy individuals (p = 0.04). In this genotype analysis of (SNP rs324981) of the NPSR1 gene, T allele was marked as risk allele with higher frequency in the obese (38%) compared to its frequency in the controls (25%). Single Nucleotide Polymorphism (SNP, rs324981) Asn107Ile of NPSR1gene, that switches an amino acid from Asn to Ile, has been found associated with increased susceptibility to obesity in Pakistani individuals. Furthermore, molecular simulation studies predicted a lower binding affinity of NPSR1 Asn107Ile variant to NPS than the wild-type consistent with the genotype studies. These molecular simulation studies predict a possible molecular mechanism of this interaction by defining the key amino acid residues. However, a significantly (p<0.0001) lower concentration of NPS was recorded independent of genotype frequencies in obese subjects compared to healthy controls. We believe that large scale polymorphism data of population for important gene players including NPSR1 will be more useful to understand obesity and its associated risk factors.

lncRNAKB, a knowledgebase of tissue-specific functional annotation and trait association of long noncoding RNA.

Long non-coding RNA Knowledgebase (lncRNAKB) is an integrated resource for exploring lncRNA biology in the context of tissue-specificity and disease association. A systematic integration of annotations from six independent databases resulted in 77,199 human lncRNA (224,286 transcripts). The user-friendly knowledgebase covers a comprehensive breadth and depth of lncRNA annotation. lncRNAKB is a compendium of expression patterns, derived from analysis of RNA-seq data in thousands of samples across 31 solid human normal tissues (GTEx). Thousands of co-expression modules identified via network analysis and pathway enrichment to delineate lncRNA function are also accessible. Millions of expression quantitative trait loci (cis-eQTL) computed using whole genome sequence genotype data (GTEx) can be downloaded at lncRNAKB that also includes tissue-specificity, phylogenetic conservation and coding potential scores. Tissue-specific lncRNA-trait associations encompassing 323 GWAS (UK Biobank) are also provided. LncRNAKB is accessible at http://www.lncrnakb.org/ , and the data are freely available through Open Science Framework ( https://doi.org/10.17605/OSF.IO/RU4D2 ).

Ca2+ signaling in T lymphocytes: the interplay of the endoplasmic reticulum, mitochondria, membrane potential, and CRAC channels on transcription factor activation.

T cell receptor stimulation initiates a cascade of reactions that cause an increase in intracellular calcium (Ca2+) concentration mediated through inositol 1,4,5-trisphosphate (IP3). To understand the basic mechanisms by which the immune response in T cells is activated, it is useful to understand the signaling pathways that contain important targets for drugs in a quantitative fashion. A computational model helps us to understand how the selected elements in the pathways interact with each other, and which component plays the crucial role in systems. We have developed a mathematical model to explore the mechanism for controlling transcription factor activity, which regulates gene expression, by the modulation of calcium signaling triggered during T cell activation. The model simulates the activation and modulation of Ca2+ release-activated Ca2+ (CRAC) channels by mitochondrial dynamics and depletion of endoplasmic reticulum (ER) store, and also includes membrane potential in T-cells. The model simulates the experimental finding that increases in Ca2+ current enhances the activation of transcription factors and the Ca2+ influx through CRAC is also essential for the NFAT and NFκB activation. The model also suggests that plasma membrane Ca2+-ATPase (PMCA) controls a majority of the extrusion of Ca2+ and modulates the activation of CRAC channels. Furthermore, the model simulations explain how the complex interaction of the endoplasmic reticulum, membrane potential, mitochondria, and ion channels such as CRAC channels control T cell activation.

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.

Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling.

Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for stable Ca(2+) handling within the cell and this novel mechanistic model provides insight into the molecular signaling characteristics that help maintain overall Ca(2+) stability even under the conditions of high SR Ca(2+) leak during CPVT. Finally, this model should provide tools for investigators to examine normal and pathological Ca(2+) signaling characteristics in the heart.

Modeling Local X-ROS and Calcium Signaling in the Heart.

Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca2+ spark rate in a process called X-ROS signaling. The increase in Ca2+ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca2+]i-sensitive RyR2 open probability increases, and the Ca2+ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca2+ sparks, and the myoplasmic calcium concentration ([Ca2+]i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca2+ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca2+ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca2+ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca2+ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not currently obtainable through experimental means and thus provides a framework for future X-ROS signaling experiments.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release.

AIMS: In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. METHODS AND RESULTS: Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca(2+) release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca(2+) sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. CONCLUSION: In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca(2+) release, increasing the likelihood of propagating Ca(2+) release.

Calcium Sparks in the Heart: Dynamics and Regulation.

Calcium (Ca2+) plays a central role in the contraction of the heart. It is the bi-directional link between electrical excitation of the heart and contraction. Electrical excitation initiates Ca2+influx across the sarcolemma and T-tubular membrane that triggered calcium release from the sarcoplasmic reticulum. Ca2+sparks are the elementary events of calcium release from the sarcoplasmic reticulum. Therefore, understanding the dynamics of Ca2+sparks is essential for understanding the function of the heart. To this end, numerous experimental and computational studies have focused on this topic, exploring the mechanisms of calcium spark initiation, termination, and regulation and what role these play in normal and patho-physiology. The proper understanding of Ca2+ spark regulation and dynamics serves as the foundation for our insights into a multitude of pathological conditions may develop that can be the result of structural and/or functional changes at the cellular or subcellular level. Computational modeling of Ca2+ spark dynamics has proven to be a useful tool to understand Ca2+ spark dynamics. This review addresses our current understanding of Ca2+ sparks and how synchronized SR Ca2+ release, in which Ca2+ sparks is a major pathway, is linked to the different cardiac diseases, especially arrhythmias.

The Phase Lag between Agonist-Induced Oscillatory Ca2+ and IP3 Signals Does Not Imply Causality (December 2015).

Activated phospholipase C (PLC*) generates 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) from phosphatidyl inositol (PIP2). The DAG remains in the plasma membrane and co-activates conventional protein kinase C (PKC) with Ca2+. We have developed a mathematical model for the activation of the Ca2+-dependent PKC and its negative feedback on phospholipase C (PLC) and coupled it to the De Young-Keizer model for IP3 mediated Ca2+ oscillations. The model describes the cascade of reactions for the translocation of PKC to plasma membrane, and simulates activation of Ca2+ and diacylglycerol (DAG) oscillations. The model demonstrates that oscillations in Ca2+ and DAG are possible with or without a positive Ca2+ feedback on phospholipase C consistent with experiment. In many experimental studies, the timing of the peaks of the Ca2+ and IP3 oscillations have been used to suggest causality, i.e. that the IP3 oscillations cause the Ca2+ oscillations. The model is used to explore this question. To this end, the positive and negative feedback between Ca2+ and IP3 production are modulated, resulting in changes to the phase lag between the peaks in [Ca2+]cyt and [IP]cyt. The model simulates a possible experimental protocol that can be used to differentiate whether or not the positive feedback of Ca2+ on PLC is needed for the oscillations.