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.

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.

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.

Superresolution modeling of calcium release in the heart.

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca(2+)) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca(2+) from the sarcoplasmic reticulum (SR). The Ca(2+) leak out of the SR is an important process for cellular Ca(2+) management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca(2+) spark. Here, we present a detailed, three-dimensional model of a cardiac Ca(2+) release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca(2+) sparks and robust Ca(2+) spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca(2+) spark and nonspark-based SR Ca(2+) leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca(2+)-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca(2+) leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca(2+) spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca(2+) release properties due to variations in inter-RyR coupling via local subspace Ca(2+) concentration ([Ca(2+)]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction.

RATIONALE: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca(2+) release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). OBJECTIVES: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. METHODS AND RESULTS: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca(2+) release and action potential prolongation. CONCLUSIONS: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca(2+) release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

The connection between inner membrane topology and mitochondrial function.

The mitochondrial inner membrane has a complex and dynamic structure that plays an important role in the function of this organelle. The internal compartments called cristae are created by processes that are just beginning to be understood. Crista size and morphology influence the internal diffusion of solutes and the surface area of the inner membrane, which is home to critical membrane proteins including ATP synthase and electron transport chain complexes; metabolite and ion transporters including the adenine nucleotide translocase, the calcium uniporter (MCU), and the sodium/calcium exchanger (NCLX); and many more. Here we provide a brief overview of what is known about crista structure and formation, and discuss mitochondrial function in the context of that structure. We also suggest that mathematical modeling of mitochondria that incorporates accurate information about the organelle's internal architecture can lead to a better understanding of its diverse functions. This article is part of a Special Issue entitled 'Calcium Signalling in Heart'.

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.

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.

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.

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.

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.

Dynamics of calcium sparks and calcium leak in the heart.

We present what we believe to be a new mathematical model of Ca(2+) leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca(2+)-release units, each containing a cluster of stochastically gating Ca(2+) channels (RyRs), whose biophysical properties (e.g., Ca(2+) sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca(2+)-release properties, and shows how this balances reuptake by the SR Ca(2+) pump. Simulations reveal that SR Ca(2+) leak consists of brief but frequent single RyR openings (~3000 cell(-1) s(-1)) that are likely to be experimentally undetectable, and are, therefore, "invisible". We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca(2+)), and occasionally lead to the generation of Ca(2+) sparks (~130 cell(-1) s(-1)). Furthermore, this physiological formulation of "invisible" leak allows for the removal of the ad hoc, non-RyR mediated Ca(2+) leak terms present in prior models. Finally, our model shows how Ca(2+) sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.

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.

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.

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.

Predicting local SR Ca(2+) dynamics during Ca(2+) wave propagation in ventricular myocytes.

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca(2+)) wave propagation, and 2), speed of Ca(2+) diffusion within the SR. Ca(2+) waves are initiated when a spontaneous local SR Ca(2+) release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca(2+) diffusion constant in the SR (D(Ca,SR)) severely complicates our understanding of whether dynamic local changes in SR [Ca(2+)] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca(2+)] during Ca(2+) waves. Simulations have investigated how dynamic local changes in SR [Ca(2+)] are influenced by 1), D(Ca,SR); 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca(2+) pumps; 4), SR Ca(2+) pump dependence on cytosolic [Ca(2+)]; and 5), the rate of transfer between network and junctional SR. Of these factors, D(Ca,SR) is the primary determinant of how release from one RyR cluster alters SR [Ca(2+)] in nearby regions. Specifically, our results show that local increases in SR [Ca(2+)] ahead of the wave can potentially facilitate Ca(2+) wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca(2+)] are possible during SR Ca(2+)release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca(2+)](SR).

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.