Intracellular Na (Na i ) elevation is a hallmark of the ischemic and failing heart – pathologies in which both acute and chronic metabolic remodeling occurs. Increased Na i is known to decrease mitochondrial calcium (Ca mito ) via enhanced Na/Ca mito exchange, potentially compromising ATP mito production leading to metabolic dysregulation. We aimed to examine whether acute (ouabain+blebbistatin) and chronic myocardial Na i load (PLM 3SA transgenic mouse) is causally linked to metabolic remodeling and if pathological hypertrophy (pressure overload) shares a common Na-mediated metabolic ‘fingerprint’. 23 Na, 31 P and 13 C nuclear magnetic resonance spectroscopy (NMR) was performed in retrogradely perfused mouse hearts followed by 1 H NMR metabolomic profiling, mass spectrometry and in silico modelling. Na i overload (acute, chronic, pathological) resulted in common metabolic perturbations: switch in substrate oxidation from fatty acid to glucose and altered steady-state metabolite concentrations (glycolytic, Krebs cycle, anaplerotic). Inhibition of Na/Ca mito exchange by CGP37157 during both acute and chronic Nai load ameliorated the metabolic changes. In silico modelling predicted increased metabolic fluxes (TCA cycle, OXPHOS, glycolysis, glucose oxidation) at the expense of metabolic coupling (glycolysis/glucose oxidation). Neither acute nor chronic Na i elevation resulted in energetic impairment (PCr/ATP) suggesting an adaptive response. Therefore, elevated Na i leads to complex adaptive metabolic alterations preceding energetic and functional impairment. Early prevention of Na i overload or reversal of the metabolic remodeling (Na/Ca mito inhibition) could potentially ameliorate the origin of metabolic dysregulation in cardiac hypertrophy and failure. These changes constitute a common metabolic ‘fingerprint’ in response to Na i elevation which appear to be independent of its aetiology or duration.
Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting 1–2% of the general population. The incidence of AF is expected to double in ~40 years. AF leads to rapid ventricular beating thereby promoting heart disease, underlies 20% of all strokes and increases death rates by 2-fold. While AF is fundamentally an electrical disorder associated with electrical re-entry, changes in tissue refractoriness, conduction block and triggered activity which collectively is called electrical remodeling, AF is also associated with changes in tissue structure and composition which also contribute in a complex manner to electrical changes and is referred to as structural remodel. Many conditions promote AF (i.e. heart disease, sleep apnea, hypertension, diabetes, endurance sport/exercise). A common feature of most conditions promoting AF is an elevated venous/atrial filling pressures which causes atrial stretch, a powerful stimulus for the activation of signaling pathways linked to hypertrophy, fibrosis, oxidative stress and inflammatory cell infiltration.
Results: Intense endurance exercise in mice for 6 weeks leads to increased atrial pressures, atrial hypertrophy, AF inducibility, fibrosis and inflammatory cell infiltrates, while having only positive physiological effects on ventricles. The adverse atrial changes with exercise were prevented by TNFα inhibition, without affecting the beneficial effects on the ventricles. Pharmacological TNFα inhibition commencing after 3-weeks of exercise did not reverse the pathological atrial remodeling. Bioinformatic analyses of RNA-seq results in atria and ventricles, with and without TNFα inhibition, revealed far more transcriptomic remodeling in atria compared to ventricles. Moreover, TNFα-mediated atrial (but not ventricular) remodeling was associated with changes in mechanotransduction-associated pathways (i.e.focal adhesion, integrin and cell-cell adhesion).
Conclusion: We conclude that intense endurance exercise leads to mechanical stretch of atria which is associated with atrial-specific, TNFα-dependent adverse structural remodeling, inflammation and increased AF vulnerability as well as the activation of mechanosensitive pathways.
Recently both 3D high resolution and functional studies in muscle cells have revealed a tightly coupled mitochondria reticulum (MR) to rapidly distribute potential energy, in the form of the mitochondrial membrane potential (MMP), throughout skeletal muscle and heart cells. Herein the structural aspects of the MR are described using 3D Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and presented in muscle cells. A large portion of the MR conductivity is dependent on direct mitochondrial matrix continuity while in some regions of the muscle the connectivity is proposed to occur via poorly characterized electron dense regions (EDR) between adjacent mitochondria. Using a photo-activated mitochondrial uncoupler to regionally perturb the MMP, we have demonstrated that large regions of the MR are electrically coupled via a shared matrix as well as EDR structures between the mitochondria. In murine skeletal muscle cells a large fraction of the mitochondrial volume is located in regions close to capillary indentations in the cell structure. These embedded capillaries are surrounded by large pools of mitochondria near the plasma membrane that have narrow tubes which run along the I-Bands (I-Band Mitochondria Segments (IBMS)) deep into the muscle cell. It has been proposed that these IBMS serve to distribute the MMP from the large sub-sarcolemma mitochondrial pool to the more central ATP-consuming myofibril region of the muscle cell. Consistent with this notion was the observation that there is a 3-fold enhancement of MMP generating oxidative phosphorylation complex in comparison to MMP utilizing ATP synthesis enzymes in the periphery of the muscle cell when compared to central regions near the muscle ATPase activity. In cardiac cells, no IBMS exist and the coupling is exclusively through large mitochondria structures and numerous EDR connections. These data are consistent with a mitochondria reticulum in muscle cells that couples large numbers of mitochondria together providing a rapid and uniform potential energy source throughout the cell to support ATP production.
The “Physiome” is the quantitative description of the functional behavior of the physiological state of an individual of a species. Physiome Projects were initiated in the early 90's, and became defined as a set of practical scientific efforts at the Znamenka Physiome Conference in 1997, a satellite of the 1997 IUPS Congress in St. Petersburg, Russia. There have been multiple Physiome meeting held internationally since then, including two on the Endtheliome, today's meeting continues the followup series from 1999 to the present targeted on quantitative cardiac physiology, mainly electrophysiology and mechanics, from channels and molecular motors to integrated cardiac contraction in vivo. This Twentieth Anniversary meeting broadens the focus to recognizing metabolism and energetics as central to the normal functioning of the heart. From the cell-centric view, the cell is the organizing element: it initiates transcription, guides the positioning of the transcribed proteins, modulates proteolysis and autophagy, and more generally speaking, regulates or remodels pathway fluxes and structural features. The Cardiac Physiome must further define growth and development, the mechanisms of maintaining the whole heart in the “milieu interieure” of the living organism, in which the organs serve each others sustenance. To integrate one ideas and understandings into self-consistent frameworks, mathematical models serve, ever more exactly though imperfectly, as “working hypotheses” around which we design experiments, teach principles, develop and test drugs, and explore therapies. The models, ever developing and never finalized, comprise the products of the Physiome Projects: they are stepping stones on the pathway of science.
Reproducing mathematical models should be a finite, completable task that will put models at the fingertips for others to test, refute and build upon. The experience of archivists of published models is that almost none of their first 1000 could be defined in computer code for distribution without going back to the author for correction or clarification. Following the stages of idea formulation, data assessment, model development with alternatives and such, modern modeling processes center on VVUQ: Verification, Validation, and Uncertainty Quantification. Verification is a mechanical exercise: to prove that the mathematical solutions are correct. Validation requires much more judgment: how many, and what kind of data sets are adequate? How well must the model solutions fit the data? Are the residuals non-random? UQ, less well defined, has three phases: (1) Parameter uncertainty estimation from the Hessian or from MonteCarlo assumes independence of parameters, though most multiscale models exhibit correlations among parameters. New statistical methods are needed. (2) Input uncertainty (the Milieu = all outside influences) need assessment. (3) Model structural uncertaintydemands that there be alternative models, perhaps at the submodel level. Together these uncertainties determine the accuracy of the model's predictions. Several groups have under development simulation analysis systems that may serve as Packages for Model Reproducibility. The Package should include the ideas, the rationale, the experimental data, the assumptions, the model (hypothesis) equations, the exact code, followed by all the VVUQ evidence PLUS the predictions, alternative hypotheses, and the designs of experiments to test the model's scientific hypotheses. Embrace open source science.
Calcium ion concentration modulates the function of several mitochondrial enzymes. Specifically, the kinetic
operations of the decarboxylating dehydro-genases pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase are all affected by [Ca2+]. Previous studies have shown that, despite its ability to affect the function of specific dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro or in vivo in the heart. We hypothesize that, rather than contributing to respiratory control, [Ca2+] plays a role in contributing to fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates (carbohydrates, fatty acids, and ketones) to synthesize ATP aerobically in the living cells. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl-L-carnitine in the media and at various calcium concentrations and ADP infusion rates.
Stoichiometric analysis of the data reveals that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both the rate of ADP infusion and to the calcium concentration. Under low-flux (leak state) conditions and with zero added Ca2+, β-oxidation provides roughly 70% of acetyl-CoA for the citrate synthase reaction, with the rest coming from the pyruvate dehydrogenase reaction. With both increasing rates of oxidative ATP synthesis and with increasing [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate. The effects of ATP synthesis load and [Ca2+] are shown to be interdependent.
The energetic status of the myocardium is compromised in decompensated hypertrophy in the failing heart, with the chemical energy (in the form of the ATP hydrolysis potential) available for the heart to do work diminished compared to normal. Using multi-scale computer models to interpret data from humans and animal models of cardiac decompensation and heart failure, we have developed two novel hypotheses to guide our investigations of how the biochemical/metabolic state of the heart in heart failure affects the mechanical pumping of the heart: (1.) Diminished cytosolic ATP and increased inorganic phosphate (associated with impaired energy metabolism and depletion of cytoplasmic adenine nucleotides) impairs the mechanical function of the heart; and (2.) By blocking purine degradation pathways that may be overactive in the chronically stressed and/or periodically ischemic myocardium, we can increase/restore the nucleotide pool and protect the heart against mechanical dysfunction and failure. Testing these hypotheses using a combination of genetic and surgical models, and computer models, our studies point to the potential promise of whole new classes of pharmacological targets associated with purine nucleotide dephosphorylation, deamination, degradation, and transport.
Accurate single-cell cardiac models of drug behavior drive the ECG and other electrical behaviors observed in
in-silico tissue level multiscale cardiac models. Typically, these models are constructed by trained professionals
over weeks and months, making it impractical to tailor simulations to a particular drug for a high-throughput screen.
We have developed a new system to automate and accelerate this model creation process. Given time varying experimental
data (i.e. from patch-clamp electrical experiments) and a differential equation with free parameters (i.e. rate
constants), our system finds values for the free parameters that reproduce the input experimental data. In the future,
we envision that our system for automated single-cell model creation will enable high-throughput drug-specific
Growing adoption of systems modeling approaches in understanding health and disease necessitates flexible and extensible computational tools. Complex physiological models are often used to run computationally-intensive simulations such as for mechanistic hypothesis testing and selection of the right pathway/target for developing treatments. To address these needs, computational tools should facilitate efficient construction and automation of such analyses. We develop SimBiology to support systems modeling and analysis, focusing on applications in drug discovery and development.
SimBiology includes functions and capabilities to perform common tasks in systems modeling, such as simulation to predict system behavior, sensitivity analysis to identify significant biological pathways, and parameter estimation to fit models to data. We present the application of SimBiology and MATLAB to systems modeling with various case studies. We also show how computationally expensive simulations of these models can be run in parallel on multicores, on clusters, or cloud for improved performance. We will also explore acausal modeling capabilities with Simscape.
To support the reuse and reproducibility of biosimulation models, authors must not only share the mathematical details of their models, but also the semantics: they must formally identify the properties and entities that are represented in their model. Our group has developed SemGen, a software tool enabling the association of parameters and variables used in a biosimulation model with semantic biological information from structured ontologies.
Models encoded in several exchange formats can be loaded into SemGen and annotated to facilitate model visualization, comparison with other models and integration to form new models. This corpus of annotated models in itself is a valuable resource embodying not only biological connectivity but quantitative descriptions of biological processes. This is our vision for the Physiome Knowledge Base (PKB) initially focusing on physiological biosimulation models.
To bridge the plethora of available biomodeling languages and platforms, SemGen and PKB are based on the Ontology of Physics for Biology (OPB) — a formal, language-free, semantic model of engineering systems dynamics that we have developed to annotate and interpret the biophysical content of simulation model code. OPB includes class taxonomies for physical properties (e.g., force, flow rate, conductance), dynamical dependencies (e.g., Hooke's law, Ohm's law) and biodynamical domains (e.g., chemical kinetics, fluid flow, thermodynamics) that are relevant to biosimulation modeling. It is a member of a publicly available family of biomedical ontologies developed in the "web ontology language" (OWL) that enforces strict logical rules and provide software tools to validate and interpret knowledge structures such as the PKB.
The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative promotes the coupling of in vitro experimental assays with theoretical models of cardiomyocyte electrophysiology and ion handling to assist in the prediction of cardiac torsadogenic drug risk. CiPA proposes the use of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) for experiments however the recommended model of cardiomyocyte electrophysiology and ion handling, the O’Hara-Rudy model (ORd), was developed to simulate electrophysiology of human adult ventricular cardiomyocytes (hAVCMs). To reconcile this mismatch, we have developed an electrophysiology model of the hiPSC-CM by incorporating differences in gene expressions of selected ion channels, exchangers, receptors and pumps in iPSC-CMs against those found in hAVCMs. The hiPSC-CM model developed here recapitulates a ventricular-like action potential along with an elevated resting membrane potential (-65 to -70 mV). The elevated resting membrane potential matches experimental data and the hiPSC-CM model shows Na+ channels in an inactivated state with the overexpression of L-Type Ca2+ channel current driving AP depolarization. This approach can be used on any hiPSC-CM cell line where expression data has been obtained and replaces background currents for K+ and Na+ in the ORd model with the ultrarapid K+ channel and hyperpolarization activated K+/Na+ channel currents. With this hiPSC-CM model, variation in two different hiPSC-CM cell lines are matched to experimental data. In addition, in silico combination blocking of Na+, L-type Ca2+ and rapid delayed rectifier K+ channels shows that variability across lines must be considered when using these in vitro assays for testing of proarrhythmic risk.
The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. This macroscale heterogeneity has been considered when building finite element models of the left ventricle, and while these models can accurately recapitulate systolic ventricular strains, they tend to exhibit unrealistically slow relaxation rates. We hypothesized that poor relaxation in such models could be due to the absence of realistic microscale heterogeneity, that is, cell-to-cell variations in myocyte properties. In the absence of specific data on microscale myocyte heterogeneity, we designed experiments to quantify the variance in mechanical properties among cells from the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free wall of adult rats, and treated with collagenase to isolate individual myocytes. Cardiomyocytes were then characterized by measuring sarcomere length changes and calcium transients during electrical stimulus. Variance of RT50, the time from peak sarcomere shortening to 50% re-lengthening, was assessed in each cell population. By starting with a full (~7 mm) longitudinal strip of myocardium, and then isolating cells from progressively shorter strips, we were able to estimate the myocardial strip volume below which regional variation vanished and only microscale heterogeneity or variance remained. Our experiments suggest that there is a minimization of variance below a tissue volume of 6 mm^3. The standard deviation of RT50 within this myocardial volume was 14.7% of the mean, which may have implications for how active contractile properties within the heart are represented.
Calcium signals coordinate contraction of the heart with each heart beat. Intracellular calcium signals arising from hypertrophic stimulation also regulate growth of heart cells. Given that these different calcium signals appear to coincide in the cell, how do they not interfere with one another? I will discuss our recent attempts to address this issue by constructing data-driven mathematical models of calcium signalling, from individual ion channels up to cell-level calcium dynamics. This forms part of a broader initiative in cardiac cell systems biology to understand the interactions between cellular signaling, mechanical and metabolic pathways in the heart in health and disease.
We present an energy-based approach to modelling biochemical reactions and pathways using the engineering-inspired bond graph methodology. We identify several advantages to this approach, using examples from heart cell electrophysiology and bioenergetics. The overall aim of our work is to present a framework for modelling and analysis of biomolecular reactions and processes which considers energy flows and losses as well as mass transport.
Reactive oxygen species (ROS) play an important role in cell signaling, growth and immunity. However, when produced in excess, they are toxic to the cell and lead to a myriad of pathologies, including organ system dysfunction, cardiovascular disease, hypertension, diabetes, and aging. A major source of ROS in many cells is the family of NADPH oxidases (NOXs) on the plasma membrane, comprising of membrane and cytosolic components. NOX2 is among the most widely expressed and studied of NOX isoforms. Although details of NOX2 enzyme structure, including description and localization of the subunits required for NOX2 assembly and activation are well elucidated experimentally, there is a lack of a quantitative understanding of the kinetic regulation of the assembly of subunits and their relative contributions towards NOX2 activation and ROS generation. Specifically, there has been no study to-date which has provided an integrated understanding of NOX2 function. To that end, we have developed here a thermodynamically-constrained mechanistic mathematical model based on diverse published experimental data sets on the catalytic action (assembly, activation and ROS production) of NOX2 in cell-free and whole-cell assay systems. The model incorporates (i) random rapid equilibrium binding mechanism for NOX2 assembly and activation, and its regulation by guanine nucleotides (GTP and GDP) and individual cytosolic subunits enhancing the binding of other subunits in replicating the subunits concentration-dependent ROS production; (ii) thermodynamics of electron transfer from substrate NADPH to molecular O2 through different redox centers, (iii) bimodal dependence of NOX2 activity upon pH variation; and (iv) distinct inhibitory effects of different drugs. In a nutshell, the present study provides the first unified hypothesis on NOX2 function, integrating different regulatory mechanisms into a mathematical model, which enables simulations of diverse available data sets with a unique set of model parameter values. The model provides a mechanistic framework to investigate the role of NOX2 enzyme in ROS production regulating diverse cellular mechanisms (e.g. NOX2-mitochondria crosstalk) in health and disease.
Increasingly we see an expectation of computational approaches (including AI) to identify previously unforeseen patterns and to inform existing human decision making. The prediction of cardiac safety liability is no exception to this trend, and the Comprehensive in-vitro Proarrhythmia Assay (CiPA) initiative is one such example. However, whilst the strategy for using high-throughput in-vitro data in computational models for calculating proarrhythmic potential is a bold and welcome approach, it is not without risk. One such risk-factor is how we should evaluate the computational models to decide whether they are fit-for-purpose (or at least no worse) in deciding about compound/drug progression. Categorisation of the proarrhythmic potential of a set of well characterised drugs is one such example when used to calibrate and validate the computational model. In this study we show how we have taken a parallel approach to consider the proarrhythmic potential of a series of known drugs in order to offer a more quantitative basis for risk assessment based on real world observation of cardiac adverse events. We demonstrate how this novel use of real world data correlates with previous risk categorisation and discuss the implications for model validation.
There has been a resurgence of interest for the field of cardiac metabolism catalysed by the increased need for new therapeutic targets for patients with heart failure. Traditionally, the focus of research in this area has been on the impact of substrate selection – carbohydrates vs. fatty acids - for mitochondrial oxidative energy metabolism. The use of recently emerging metabolomic technologies - which aim at systematically measure all low-molecular weight compounds within a biological system - has provided some novel insight into the global metabolic perturbations prevailing in several cardiovascular diseases. Acylcarnitines (ACs) are among metabolites that have been the focus of many recent metabolomics-based studies, particularly in human heart failure. Profiling of circulating ACs has commonly been used for diagnosis of inborn errors of metabolism, particularly mitochondrial fatty acid oxidation (FA) defects. Beyond being proxies of fatty acid metabolic dysregulation, ACs - primarily long-chain ACs (LACAs) – are, however, increasingly being recognized as “actors”, modulating cell functions, as well as being linked to adverse cardiac events such as arrhythmias. This presentation aims to provide an overview of metabolic pathways generating ACs and of studies reporting elevated circulating levels of ACs, particularly LCACs, in human heart failure. It will also discuss proposed molecular mechanisms for the potential adverse effects of LCACs on cardiac function.
When a coronary artery becomes occluded, flow of oxygenated blood to the surrounding heart muscle is compromised and the cardiac myocyte muscle cells suffer severe ischemia. In a recent paper [J. Biol. Chem. (2017) 292(28) 11760 –11776] we provide a computational model that follows the state of the myocytes following oxygen deprivation. A surprising result is that the myocytes can survive for substantial periods of time if there is some residual oxygen available, as from collateral circulation. Experiments have shown that the event of reperfusion causes the death of a significant fraction of the ischemic myocytes, and this result is termed the ischemic/reperfusion problem. Recent advances in understanding the detailed metabolism of the mitochondria suggest that it may be possible to extend the computational model to follow the highly nonlinear reperfusion events where reactive oxygen species are formed and competing demands for ATP can jeopardize the cells’ ability to function.
The more recent studies of human pathologies have essentially revealed the complexity of the
interactions involved at the different levels of integration in organ physiology. Integrated organ thus reveals functional properties not predictable by underlying molecular events. It is therefore obvious that current fine molecular analyses of pathologies should be fruitfully combined with integrative approaches of whole organ function. It follows that an important issue in the comprehension of the link between molecular events in pathologies and whole organ function/dysfunction is the development of new experimental strategies aimed at the study of the integrated organ physiology.
By combining the principles of control analysis with non-invasive 31P NMR measurement of the energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (for Modular Control and regulation Analysis), an integrative approach designed to study in situ control and regulation/dysregulation of cardiac energetics during contraction in intact beating perfused isolated heart (Diolez et al., Am J Physiol Regul Integr Comp Physiol 293(1):R13–R19, 2007). MoCA can potentially be used not only to detect the origin of the defects associated with a pathology, but also to provide the quantitative description of the routes by which these defects - or drugs - modulate global heart function, therefore opening therapeutic perspectives.
We will present selected examples of the applications to isolated intact beating heart and discuss how this approach could be applied to cardiac energetics under clinical conditions to help in the future for diagnosis and therapeutics follow-up in human heart pathologies.
The ultra-rapid delayed-rectifier K+ current (IKur), carried by the atrial predominant (vs. ventricles) alpha subunit KV1.5, has been studied as a promising target to treat atrial fibrillation (AF). However, while numerous KV1.5-selective compounds have been screened in vitro and in animal models of AF, evidence of antiarrhythmic efficacy in human clinical trials is lacking, perhaps because preclinical assessment of candidate drugs overly relies on steady state drug concentration response curves rather than accounting for channel conformational state specificity and kinetics of drug binding. Here, we simulated a Markov-type model of IKur gating and drug-channel interaction within our comprehensive atrial cell model to reveal the ideal binding properties of IKur inhibitors that maximize AF-selectivity in normal sinus rhythm (nSR) and chronic AF (cAF). Specifically, we identified drugs exhibiting anti-AF properties at fast-pacing rates (prolongation of effective refractory period, ERP), while having little effect during normal heart rhythm (limited prolongation of action potential duration, APD). We also found that despite being downregulated in our simulations (by 50%), IKur contributes more prominently to APD and ERP in cAF than in nSR, and block of IKur in cAF has less cardiotoxic effects and increased efficacy. We propose that our in silico strategy can be combined with in vitro and in vivo assays, as championed by the Comprehensive in Vitro Proarrhythmia initiative, to identify the complex impact of IKur inhibitors at the different stages of AF-induced remodeling.
In adults, most wounds are repaired with the formation of a scar. Scar formation limits tissue functionality. For instance, after myocardial infarction, the formation of scar tissue limits the ability of the heart to pump enough blood to the rest of the body. The increased stress on the heart can lead to congestive heart failure. In contrast to adults, embryos display an outstanding ability to rapidly repair wounds, in a process driven by collective cell movements. Actin and the motor protein non-muscle myosin II become polarized in the cells adjacent to the wound, forming a supracellular cable that contracts to coordinate the movements that drive tissue repair. We showed that, in Drosophila embryos, the cable is heterogeneous, with regions of high and low actin density. Mutants in which actin is uniform around the wound display slower wound closure. However, the mechanisms by which a non-uniform distribution of actin favours rapid repair are unknown. Using laser ablation, we demonstrated that actomyosin-rich segments of the cable sustain higher contractile forces, indicating that cable contraction is non-uniform. We developed a computer model of wound repair, and we found that a heterogeneous actomyosin distribution was favourable for wound closure when myosin assembly at the wound edge was strain-dependent. To test the model prediction, we used a laser-based method to induce ectopic strain on cell boundaries in vivo, and we found that myosin accumulated in response to deformation. Using pharmacological and genetic treatments, we found that stretch-activated ion channels were necessary for rapid embryonic wound repair. Our results suggest that local heterogeneities in supracellular actomyosin networks promote faster contraction by generating mechanical signals sensed by stretch-activated channels that facilitate myosin assembly and coordinate cell behaviours.
Cardiac muscle cells are reported to contain a cell wide mitochondrial reticulum where mitochondria are interconnected in networks though intermitochondrial junctions. We have also observed that mitochondria form heterogeneous clusters within localised regions of the cell. Recent studies suggest that conduction of membrane potential through the mitochondrial networks can act as one of the dominant mechanisms to distribute energy across the cell. There are several other myofibril based metabolite diffusion systems present in cardiomyocytes which can also contribute to intracellular energy transport. What is the interplay between these different mechanisms and the heterogeneous distribution of mitochondria? We have used a finite element model of cardiac bioenergetics and force dynamics to investigate this question.
Our study indicates that diffusion of O2 is insufficient to reach interior of a cardiomyocyte in a high workload – leading to a regional hypoxia at the core of the cell. However, conduction of membrane potential from the mitochondria at the cell periphery can provide sufficient protonomotive force to the mitochondria at the cell core to sustain a uniform distribution of metabolites and force dynamics across the cell cross section. However, our study also indicates that rapid diffusion of creatine and phosphocreatine ensures that metabolites like ATP and ADP are uniformly distributed in the midst of a heterogeneous distribution of mitochondria that we observe in the cell. Thus both mitochondrial membrane potential conduction and myofibrilar diffusion systems are necessary to develop a uniform metabolic and mechanical landscape across the cross-section of a cardiomyocyte.
The importance of the microcirculation in tissue function, and in particular the role of microvascular network properties, is well known. However, detailed investigation of the effects of network structure on blood flow, blood-tissue exchange and flow regulation has received less emphasis as of late. Therefore, our research group has been using intravital videomicroscopy to determine arteriolar and venular network structure in a rat gluteus maximus (GM) muscle preparation we recently developed. We have also been working to determine how the integrated arteriolar/capillary/venular network determines and regulates blood flow. Since a main purpose of tissue blood flow is delivery of oxygen to parenchymal cells, we have begun using a computational model to investigate oxygen transport between our microvascular networks and the surrounding tissue. This work uses a discrete description of arterioles and venules coupled to a tissue model containing continuously distributed capillaries. For baseline blood flow and oxygen consumption, we present results showing the loss of oxygen from arterioles, the tissue oxygen distribution, and the uptake of oxygen by venules. We also present results for both global and local increases in oxygen consumption, with and without oxygen-dependent flow regulation. These simulation results will be tested against experimental data, with the ultimate goal of building a detailed, validated model of microvascular transport and flow regulation in the GM that can be generalized to the heart and other tissues.
Pulmonary arterial hypertension initiatTowards organs-on-a-plate and injectable tissueses a cascade of pathological events in the heart. The right ventricle (RV) endures pressure overload before it hypertrophies, leading ultimately to right-ventricular failure (RVF). The left ventricle, in contrast, suffers atrophic remodelling, as RV hypertrophy causes the septum to bow leftwards, subjecting the LV to reduced load and prompting it to undergo a reduction in mass and cavity size. Does atrophic remodeling affect the energetics, in particular the energy efficiency, of the LV? To answer this question, we studied RVF rats 6 weeks following a single injection of monocrotaline which progressively damages the endothelial cells lining the vessels of the pulmonary circulation. Experiments were performed at body temperature and were designed to measure LV mechanoenergetic performance at two physiological levels. At the ex vivo working-heart level, the LV generated a lower pressure-volume work output in the RVF group compared with the Control group, over a wide range of afterloads. The lower work output was commensurate with a lower oxygen consumption, and, hence, energy efficiency was unchanged. At the in vitro tissue level, LV trabeculae of the RVF group liberated lower heat output. Their force-length work output was not significantly lower and, hence, efficiency was unchanged. Our collective data show that in hypertrophied right-ventricular failure secondary to pulmonary arterial hypertension, the left ventricle, despite undergoing atrophy, has normal energy efficiency, over a wide range of afterloads, at both the organ level and the tissue level.
Intracranial pressure (ICP) and cerebral venous hemodynamics are significantly affected by changes in posture. Transitions from the supine to the upright posture leads to drops in ICP (when measured in the ventricular space), jugular venous collapse, and cerebral venous outflow mediated via the vertebral venous pathway. To assess the effect of posture on cerebrovascular hemodynamics, we developed a multiscale model of the arterial, venous, and cerebrospinal fluid systems, coupling a lumped parameter model of the cerebral circulation with a three-dimensional model of the jugular veins. The collapsible vessel model of the jugular vein incorporates fluid-structure interaction and large-deformation nonlinear mechanics. The nonlinear finite-element method for the vessel wall is solved by using an explicit total Lagrangian algorithm. We also use a penalty method to solve the surface contact problem between opposing parts of the vessel wall during collapse. For the blood-flow simulation, the Navier-Stokes equation is solved for incompressible flows. Simulations start during posture change from supine to standing and continue through a simulated Valsalva maneuver in the standing position. Our simulations reproduce jugular venous collapse during tilt and reopen during the Valsalva maneuver. A hysteresis pattern in the pressure-volume relation of the jugular vein is observed during collapse and reopening. In conclusion, venous collapse is a highly nonlinear process and fluid-structure interaction models may allow realistic representation of the underlying vascular biophysics. Our work is a step towards a three-dimensional biophysical model of the cerebral venous system for investigation of a variety of physiological and pathological conditions.
Pulmonary veins (PVs) are considered to trigger atrial fibrillations by inducing ectopic activities. In experiment, noradrenaline (NA) stimulation was shown to induce arrhythmogenetic automaticity in rat PV cells (PVCs). Although involvement of Ca2+ release
from sarcoplasmic reticulum (SR) was suggested to play an important role in inducing the spontaneous rhythm by activating NCX,
detailed ionic mechanisms underlying the automaticity remain to be clarified. For quantitative understanding of the mechanisms, we firstly developed a mathematical model of the PVC by integrating experimental data published previously. Secondly, bifurcation analysis was applied to an intracellular Ca2+ dynamic model, which was reduced from PVC model. It was revealed that within a certain range of total intracellular Ca2+ amount, the model had unstable equilibrium points accompanied by stable limit cycles.
These results suggested that this Ca2+ dynamic model had a potency to generate a rhythmic Ca 2+-induced Ca2+ release (CICR) in itself. Thirdly, conditions required for the development of action potentials (APs) triggered by delayed after depolarizations (DAD) induced by CICR via NCX were elucidated using the full PVC model including the reduced Ca2+ dynamic model. It was confirmed that whether AP is triggered or not is determined by the balance between the total membrane conductance and the amplitude of DADs. Lastly, the effects of NA stimulation on generation of automaticity were investigated using the full model. Functional roles of individual ionic channels and transporters in generating NA-induced automaticity in the PVC model will be discussed.
Current methods of echocardiography provide detailed information about heart wall motion during the cardiac cycle. However, the ability to interpret these data in terms of cardiac muscle properties is limited. The eventual goal of the present work is to develop theoretical models for left ventricle (LV) dynamics that allow estimation of myocardial stiffness and contractility from echocardiogram data. To reduce the computational complexity of this inverse problem, we represent LV deformation in terms of a limited number of modes. We have previously presented an axisymmetric model that describes the essential motions of the LV myocardium with three volume-conserving deformations. Here, we extend this framework to a more general set of volume-conserving deformations. The reference and deformed configurations of the LV are described using prolate spheroidal coordinates. The initial boundaries of the LV are defined by splines. We derive a family of mappings using a relatively small number (< 20) of time-dependent parameters to represent the main modes of LV deformation. These deformations are chosen to closely represent the important characteristics of LV motion, such as contraction, torsion, and elongation. Non-axisymmetric modes of deformation, such as those resulting from differences between the septal and lateral walls, are included. We construct a mechanical model that incorporates muscle fiber orientations, active and passive stresses, and surface tractions. We compare this model to the axisymmetric model to evaluate the relative importance of these additional deformation modes in determining overall cardiac function, as a step toward better quantification of heart disease.
Calcium plays a central role in mediating the contractile function of heart cells. However, calcium is also the second messenger in a wide variety of other intracellular signalling pathways, including hypertrophic signalling in cardiomyocytes. How intracellular calcium can encode several different, specific signals at once is not well understood. In heart cells, calcium release from RyRs triggers contraction. Under hypertrophic stimulation, calcium released from IP3R channels triggers dephosphorylation and nuclear import of the transcription factor NFAT, with resulting gene expression linked to cell growth. Yet this must occur on a background of rising and falling cytosolic calcium with each heart beat. We show experimentally that, after addition of IP3, there is an increase in the full width at half maximum (FWHM) of the cytosolic calcium transient in adult ventricular myocytes. Computational modelling indicates that this can be attributed solely to activation of IP3R channels within the cytosol. This work, together with a recent study which shows that dephosphorylation and nuclear import of NFAT responds to the cumulative calcium load, indicates that this increase in width may be the key signal-carrying component of the IP3-dependent hypertrophic signalling pathway in cardiomyocytes.
The associated brief abstract: Small GLP-1 peptide fragments demonstrate cardiac protection from ischemic injury. Somewhat surprisingly, isolated cell, heart and in vivo experimental models reveal that this effect is dependent on the ability of these peptides to enter and modulate the metabolism of coronary vascular smooth muscle (and endothelial cells) and not cardiac myocytes. These results identify new cell types and pathways as therapeutic targets for a condition where there remains no approved treatments.
Catecholaminergic Polymorphic Ventricular Tachycardia type 2 (CPVT2) is caused by a point mutation in the calsequestrin protein. Calsequestrin is found in the junctional sarcoplasmic reticulum (jSR) where it serves as a Ca2+ buffer and as a modulator of the cardiac ryanodine receptor (RYR2) open probability (Po). The more luminal Ca2+ it binds, the greater the sensitivity of RYR2 to be opened by subspace [Ca2+]i. The CPVT2 mutation results in an increased open probability of the RyR2, which served to increase RyR2 Po and hence the leak of Ca2+ out of the SR. Patients/animal with CPVT2 display normal cardiac function at rest, but develop a possible fatal arrhythmia during exercise (beta adrenergic stimulation). Simulations show that the increase in RyR2 open probability and the decrease in SR Ca2+ buffer that accompany the mutant calsequestrin alone are not sufficient to cause an arrhythmia under beta adrenergic stimulation due to the increased leak of Ca2+ out of the SR. Hence, there are no large spontaneous Ca2+ transients which are considered arrhythmogenic. The model demonstrates that the increases in junctional SR volume that also accompany the mutation are critical in creating the proper conditions for arrhythmia. The net result is a steep mechanical restitution curve that created the substrate for alternans which is the observed arrhythmia that accompanies CPVT2.
Exercise stress testing is increasingly used in clinical cardiac Magnetic Resonance imaging (MRI) to detect abnormal heart function. We investigated if the current precision of cardiac 31P Magnetic Resonance spectroscopic assay of myocardial energy balance is technically sufficient to detect metabolic failure during exercise stress testing in heart patients. The study was conducted in 8 healthy subjects using a state of the art 3 Tesla MR scanner equipped with 31P surface coils and spectral localization techniques commonly available from major vendors. Precision of ISIS-based estimation of the basal myocardial PCr/ATP ratio was twofold better than CSI-based spectral localization techniques (1.57 +/- 0.17 versus 1.70 +/- 0.56, respectively; mean +/- SD; acquisition time 400 s). The ISIS estimate was used to condition the basal state of a computational model of myocardial oxidative ATP metabolism (Wu et al PNAS 2009). Simulations of myocardial energy balance during exercise stress testing were performed for two pathophysiological conditions associated with heart failure: progressive creatine depletion and mitochondrial failure, respectively (Neubauer NEJM 2007). Model uncertainty was computed using a Monte Carlo approach and the ISIS assay SD. The results showed that at least a twofold improvement in 31P MRS assay precision will be needed for any added value of exercise stress testing in clinical 31P MRS investigations of metabolic homeostasis in heart failure. Advanced 31P coil technology (phased array) and stronger magnets (7 Tesla scanner) may soon put this objective within reach (www.metascan.nl).
Cardiomyopathy and exertional rhabdomyolysis are severe complications in human long chain fatty acid oxidation (FAO) disorders such as very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Failing ATP homeostasis in cardiac and skeletal muscle due to impaired FAO in combination with glycogen depletion is thought to be the underlying cause. Indeed, in vivo 31P Magnetic Resonance spectroscopic (31P MRS) examination of energy balance in quadriceps muscle of VLCADD patients performing low-intensity bicycling exercise revealed an excessive drop of phosphate potential compared to healthy controls (Diekman et al PLoS ONE 2016). Acute supplementation with ketones as alternative oxidative substrate may offer effective metabolic therapy in this and other metabolic (cardio)myopathies (Veech et al IUBMB Life 2001). Indeed, it was previously shown in a canine model that ketone infusion raised the phosphate potential in cardiac muscle in vivo (Kim et al AJP Heart 1991). Here, we report on a randomized, blinded, placebo-controlled, 2-way crossover study of the acute metabolic effects of a ketone ester drink (Cox et al Cell Metabolism 2016) on muscle energy balance during exercise in five VLCADD patients. We found that the inorganic phosphate/phosphocreatine concentration ratio in exercising quadriceps muscle was significantly lower on ketones than placebo. This result demonstrates the potential of ketone therapy targeting mitochondrial metabolism in human (cardio)myopathies including heart failure (Brown et al Nat Rev Cardiol 2017).
Cardiac excitation-contraction coupling (ECC) is characterized by a synchronous rise in intracellular Ca2+ during systole, afforded by close coupling of L-type Ca2+ channel (LCC)s on myocyte sarcolemma (SL) with ryanodine receptor (RyR) Ca2+ channels embedded on the sarcoplasmic reticulum (SR). This coupling is facilitated by invaginations of the cell membrane called transverse tubules (TTs) that align LCC-rich cell membrane with RyRs in functional units called Ca2+ release units (CRUs). Recent imaging data have revealed intricate membrane microstructure within TTs that may influence Ca2+ SL handling. The close relationship between TT structural organization and Ca2+ homeostasis implicates SL Ca2+ entry in maintaining efficient ECC, yet few studies have carefully examined the influence of the intra-TT microstructure on Ca2+ exchange between the extracellular and cytosolic regions of the cytosol. To elucidate this relation, we used finite element simulations of Ca2+ diffusion to examine signaling processes within subcellular domains defined by membrane microstructure. We discuss our findings pertaining to prevailing factors that control subcellular Ca2+ signaling microdomains, and moreover how such models can be extended to diverse signaling phenomena, including metabolism.
Dialysis is a repetitive sub-lethal ischemia1. Cardiac CT imaging has shown dialysis to alter ventricular perfusion heterogeneity, which may be related to ischemia induced loss of mechanical function. Using a model of human coronary vasculature, we investigated cause-effect relationships between blood viscosity, artery occlusion, and blood vessel radius to perfusion heterogeneity.
CT images from three patients pre- and post-dialysis were analysed. Perfusion heterogeneity in each heart was quantified using fractal dimension2. Anonymised clinical data for each patient were obtained. A bi-ventricular anatomy was generated as truncated ellipsoids3. Morphometry data in conjunction with the space filling algorithm of Beard and Bassingthwaighte4 were used to generate multiple instances of coronary vasculature networks up to a resolution of 0.07 mm (imaging resolution). Conservation and scaling laws were applied to compute biophysical variables including perfusion. The fractal dimension of small sections of the model was computed to establish the baseline perfusion heterogeneity. The model was implemented using in house C language codes. The simulations are being performed on Compute Canada HPC resources.
Among other results, an increase of fractal dimension between pre- and post dialysis CT scan data. The model indicated that a major factor is reduction of small arteriole diameter.
1C. W. McIntyre,Seminars in dialysis 23, 449 (2010). 2J. B. Bassingthwaighte, et al.,Circ Res 65, 578 (1989). 3J. C. Mercier, et al.,Circulation 65, 4962 (1982). 4D. A. Beard, et al.,Journal of vascular research 37, 282 (2000).
Careful mapping of three-dimensional (3D) cardiac structure in the micro-to-macro domain has provided an atlas for exploration of structure-function cross-talk in the heart. This laid basis for previously unthinkable progress in conceptual and computational models, inspired hypothesis formation for novel experiments, and is starting to translate into guidance for patient treatment. In order to focus on intracellular signalling, the same comprehensive spatio-temporal characterisation is needed for single cells, in the nano-to-micro domain. The spatial resolution of a variety of available techniques, from super-resolution fluorescent imaging (102 nm)3, focussed ion-beam / scanning electron microscopy (EM) (101 nm)3, and EM-tomography (100 nm)3, provides 3D insights into membranous and filamentous structures of various cardiac cells in situ over increasingly meaningful cellular volume fractions. This talk will focus on cardiac EM-tomography which, over the past eight years, has started to move from a somewhat outlandish effort (Circ Res 2009 Mar/104:787-95; J Cell Sci 2009 Apr/122:1005-13) to becoming productive for (nearly) everyday research questions (J Clin Invest 2016/126:3999-4015; PNAS 2016/113:14852-14857; PBMB 2016/121:77-84; Sci Rep 2017/17:40620; JMCC 2017/108:1-7; Biophys J 2017/113:1047-1059; Physiol Rep 2017/5: e13437; eLife 2017/6: e24662; Note: examples listed are from the first and the most recent 12-months-period of application of EM-tomography to the heart). The challenge now is to generalise available information and to include it into a structured understanding – to allow better navigation of the challenges associated with exploring the dynamically deforming and densely populated reactor we call ‘the cell’.
In silico cardiac myocyte models present powerful tools for drug safety testing and predicting phenotypical consequences of ion channel mutations but their accuracy is sometimes limited. For example, several models describing human ventricular electrophysiology perform poorly when simulating effects of long QT mutations. Model optimization represents one way of obtaining models with stronger predictive power. Using a recent human ventricular model, we demonstrate that mlinkbookmark html codeodel optimization to clinical long QT data in conjunction with physiologically-based bounds on intracellular calcium and sodium concentrations better constrains model parameters. Testing the optimized models against a database of known arrhythmogenic and non-arrhythmogenic ion channel blockers resulted in improved risk assessment. In particular, we demonstrate a reduction in the number of false positive outcomes generated by the standard model. This result underscores recent studies pointing to a role of intracellular calcium concentration as a torsadogenic risk factor. Our study highlights the need for rich data in cardiac myocyte model optimization and substantiates such optimization as a method to generate models with higher accuracy of predictions into drug-induced cardiotoxicity.
Mitochondria are essential organelles in the energy metabolism of cells. The production of ATP from the mitochondria is based on their metabolites, however, their functional changes depending on mitochondrial substrates has not been clearly described. The status of the mitochondria without substrates is also not clear. In this study, NADH, mitochondrial membrane potential (Ψm) and oxygen consumption were monitored in different set of mitochondrial metabolites such as malate, pyruvate, glutamate and succinate in the presence or absence of inorganic phosphate. Any single metabolite could not maintain mitochondrial function, except succinate, which could consume the oxygen, however, Ψm was not effectively formed. Some of combinations of the metabolites could generate mitochondrial membrane potential and oxygen consumption. However, the extents of the NADH production, Ψm formation, and oxygen consumption were different depending on the substrate combination. These results suggest that the energized mitochondria are not the same when the different substrates were used and that the effective energy metabolism and the production of ATP may require a suitable metabolites combination. To understand the mitochondrial function, it is essential to know the status of mitochondria in deenergized conditions. Our results suggested that the considerable membrane potential is remained without the substrates and the addition of Pi further hyperpolarized Ψm. Surprisingly, when we add both Pi and ATP, ΔΨm was dramatically further hyperpolarized about 50mV. In addition, FAD signal was increased which reflected FADH consumption. NADH signal was also increased, however, very small compared to FAD change. Interestingly, the addition of diazoxide (mitochondrial KATP channel opener) could inhibit Pi/ATP-induced hyperpolarization and FAD increase, but not completely. The cytosolic K+ replacement with N-methyl-d-glucamine(NMDG), glibenclamide, TEA and 5-HD could partially attenuate Pi/ATP-induced effects. Interestingly, oligomycin A almost completely abolished the Pi/ATP-induced effects. From the above results, we postulated cytosolic K+ is essential to generate Pi/ATP-induced changes. Mitochondrial KATP channel may not participate in Pi/ATP-induced changes. Somehow, F1,F0-ATPase may control the FADH/FAD conversion with K+ ion. This research was supported by a fund (NRF-2016M3C1A6936606) from NRF.
Endogenous, intramyocellular triglyceride in the heart is now understood to be a dynamic and physiologically active metabolite pool, providing long chain fatty acids as ligands for nuclear receptors, fuel for energy metabolism, and substrate for acyl intermediates that induce lipid signaling. Metabolic remodeling of the heart due to pathological stress induces maladaptive changes in LCFA metabolism and triglyceride pool dynamics which can be mediated by specific chain length and saturation of exogenous LCFA.
Scar-related arrhythmia is a major cause of sudden cardiac death worldwide. Image-based computational models for electrophysiology can help us understand and predict such arrhythmias. Our goal is to develop preclinical models using advanced MR imaging methods that efficiently probe the biophysical MR signal in chronically infarcted myocardium, and to couple these models with computer modelling to enable accurate predictions of electrical wave propagation through the heart. In this feasibility study, n=5 swine with chronic infarct and one healthy swine underwent an MRI study, followed by an X-ray guided electrophysiology study using catheter-based electrical mapping systems. For infarct imaging, we employed our custom-developed T1-mapping MR method (1x1x5mm spatial resolution). We used the MR images as input to a robust fuzzy-logic algorithm, and segmented the infarcted zone into: unexcitable dense fibrosis and slow conducting peri-infarct (i.e., mixture of viable and non-viable myocytes forming arrhythmia substrate). Using these segmented images we further built high-fidelity 3D predictive heart models, integrating the three zones: healthy myocardium, dense fibrosis and peri-infarct into computational meshes (1mm element size). Finally, we investigated the accuracy of model predictions by comparing the simulated and measured isochronal maps (i.e., depolarization time maps). Overall, results showed that our predictive T1-based heart models are sufficiently accurate; the mean absolute error between the simulated and measured depolarization times was small (~7ms for the control heart and ~10ms in average for the infarcted hearts). Future work will focus on refining the spatial resolution of the current T1 imaging method and on simulating the arrhythmia inducibility.
Adenosine (Ado) plays a critical role in regulating coronary flow, especially during hypoxia, acting on and smooth muscle cells. Ado also influences cardiomyocytes and is taken up into ATP: (1)how much plasma Ado reaches cardiomyoctyes is unclear as endothelial cells (EC) avidly take-up Ado from blood stream and interstitium; and (2) inside the cells, how much Ado degrades to uric acid (UA),or is salvaged back into ATP pools is unclear. Our goal here is to quantify the fluxes of Ado and itsmetabolites in ECs. The multiple-indicator-dilution technique (MID) was used to measure the capillarytransport and metabolism of Ado and metabolites in isolated, Krebs-Ringer perfusated guinea pig hearts. Radiolabeled [ 14 C]- adenosine, or inosine, or hypoxanthine, or xanthine was injected with [ 131 I]-albumin (intravascular reference solute) and [ 3 H] AraH or L-[ 3 H] glucose (extracellular references) as a bolus into the coronary flow; venous outflow dilution curves of the injectate and its metabolites were quantified with HPLC and LS counting. The permeabilities of each solute (via interendothelial clefts,PSg, trans-endothelial membrane, PSecl, trans-myocytes PSpc) and the metabolism in ECs and cardiomyocytes were estimated by fitting the MID curves with a general convection-diffusion-exchange-reaction model, Gentex. Results are: Intracellular sequestration of injected Ado is high compared to its derivatives (~60% of Ado vs. ~0% of UA). Similarly, PSecl is high for Ado >INo>Hx>Xa> UA (Ado:2.1 vs UA:0.12 ml/min/g). The low permeabilities help to retain purine in the cells. Endothelial Ado enters the ATP pool faster than it is lost from the cell, so transient retention is high. Plasma injectate Ado is 75% oxidized to UA, 25% retained; Hx is 45% oxidized, 55% retained, both presumably incorporated into nucleotide pools. Thus ECs actively participate in purine salvage via both Ado and Hx. Having well-defined parameters for permeabilities and metabolism for Ado and its derivatives in ECs is an essential step toward understanding the fate of endogenous Ado generated from cardiomyocytes during ischemia and reperfusion.
Cardiac muscle converts the chemical free energy obtained by oxidation of metabolic substrates into mechanical work. Overall thermodynamic efficiency is the fraction of substrate free energy that appears as work. The overall energy cost of a cardiac twitch comprises two conceptually distinct contiguous components: "Initial metabolism (I)" and "Recovery metabolism (R)". Initial metabolism funds electrical excitation, Ca2+ triggering and work by the contractile proteins, events fuelled by the free energy of ATP hydrolysis. There is sufficient ATP to fuel only a brief period of activity. Buffering of ATP by PCr and regeneration of PCr via mitochondrial ATP production underwrites sustained activity. Regeneration of PCr by ATP produced in the mitochondria constitutes Recovery Metabolism. The initial and recovery processes are coupled in series. Therefore, overall thermodynamic efficiency is the product of the efficiencies of the initial and recovery processes.
Thermodynamic efficiency cannot be measured directly, so must be inferred from mechanical efficiency, the ratio of work to overall enthalpy output. Since the difference between enthalpy and free energy arising from the oxidation of either carbohydrates or fatty acids is negligible, overall thermodynamic efficiency can be safely approximated by overall enthalpy production. Rescaling the free energy of ATP hydrolysis (60 kJ/mol) by the enthalpy of PCr hydrolysis (34 kJ/mol), and adopting our measured value of activation enthalpy (20% of total energy expenditure), allows us to conclude that the thermodynamic efficiency of mitochondrial oxidative phosphorylation of either fats or carbohydrates is approximately 0.8 while that of cross-bridge cycling is approximately 0.24.
The mitochondrial calcium uniporter (MCU) relays cytosolic calcium transients to mitochondrial metabolic machinery. Increasing cardiac workload by inotropes preferentially stimulates glucose oxidation, compared to fatty acids oxidation, due to stimulation of intramitochondrial Ca2+- sensitive dehydrogenases including pyruvate dehydrogenase (PDH). However, it is not clear what effect increasing workload has on cardiac energetics and cardiac function if mitochondrial Ca2+ levels are decreased. We examined this in hearts from inducible cardio-specific deleted MCU (MCU-/-) mice. Isolated working hearts from MCU-/- and MCUfl/fl control mice were perfused with 5 mM glucose, 0.8 mM palmitate, 3% albumin, with or without insulin (100 μU/ml) or isoproterenol (10nM). Interestingly, MCU-/- hearts showed higher cardiac work compared to control hearts, and were not energy-starved, as they displayed rates of glucose and fatty acid oxidation comparable to control hearts. Isoproterenol treatment of MCU-/- hearts increased glucose oxidation similar to control hearts, although palmitate oxidation increased to a greater extent in MCU-/- hearts compared to controls (793±60 vs. 558±55 nmol.g dry wt-1.min-1, p < 0.05, respectively), resulting in a greater reliance on fatty acid oxidation for ATP production. The rise in fatty acid oxidation correlated with lower levels of malonyl CoA, an endogenous fatty acid oxidation inhibitor, and to a stimulatory increase in acetylation of 3-hydroxyacyl CoA dehydrogenase, a key enzyme of the fatty acid oxidation pathway. These results suggest that low mitochondrial Ca2+ does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation.
Approximately 6.5 million Americans are affected by heart failure, a condition where the heart is unable to meet the blood and oxygen demands of the body. Pathophysiological changes that occur during heart failure include remodeling of the myocardium and alterations in the metabolic state that are associated with the depletion of key metabolic pools in the heart. Based on multi-scale computer simulation of mechanical-metabolic interactions in the myocardium, we predict that depletion of the myocardial adenine nucleotide pool in heart failure impedes the ability of oxidative phosphorylation to supply ATP at the hydrolysis potential necessary for proper physiological function of the heart. Furthermore, we hypothesize that the resulting changes to the phosphate metabolite levels impair mechanical function of the heart, and contribute to the heart failure phenotype. To test these hypotheses, transverse aortic constriction (TAC) surgery was performed on 3-week-old Sprague Dawley rats to induce heart failure. Mechanical function of the heart was measured by echocardiography at several time points post-surgery and, after 15 weeks, hearts were excised to measure mitochondrial oxidative capacity and myocardial cytosolic metabolites. We observe reductions in fatty acid and carbohydrate oxidative capacities and adenine nucleotide pool levels that correlate with impaired mechanical function. Furthermore, with decreased ejection fraction there is a decrease in the magnitude of the ATP hydrolysis potential. Taken together these results suggest that depletion of the adenine nucleotide pool and reduction in mitochondria oxidative capacity both contribute to metabolic and mechanical dysfunction in this model. Future studies include investigating whether cardiac function can be improved in heart failure by inhibiting or reversing adenine nucleotide degradation and efflux. Preliminary studies on an inducible ventricular 5' nucleotidase knockout suggest that by inhibiting adenine nucleotide pool depletion, the energetic status and mechanical function of the myocardium in heart failure may be improved.
Cardiotoxicity is one of the major safety concerns that lead to a discontinuation of the development process as well as the market use of drugs. Despite the recognized role of current preclinical toxicology methods based on hERG ion channel inhibition or prolongation of the QT interval, they mostly focused on electrophysiological changes caused by drugs and frequently failed to predict contractile and structural heart toxicity related to a long-term drug exposure. To develop a method that could predict those cardiotoxic-related changes and potential safety of drugs, we analyzed the gene expression profiles of 30 safe and 23 cardiotoxic drugs. For this purpose, we used FAERS and SIDER databases of side effects and the literature to collect the list of compounds with cardiac side effect and matching safe compounds. After that, drugs were linked to their gene expression profiles in the DToxS and Connectivity map databases. Differential gene expression and pathway analysis were performed to compare signatures of cardiotoxic and safe drugs and to preprocess the data to train a set of machine learning classifiers. In this study, we demonstrated that cardiotoxic drug signatures are diverse, which reflects the heterogeneity of cardiotoxic mechanisms. We also proposed some of the additional mechanisms of cardiotoxic drugs, such as inhibition of ion channel trafficking for a tyrosine kinase inhibitor imatinib. Furthermore, we achieved test set sensitivity (recall) of 90% and specificity (precision) of 82% in discrimination cardiotoxic and non-cardiotoxic drugs. These techniques could, therefore, complement current preclinical and clinical practice for drug safety testing.
Understanding and predicting how ion currents contribute to cellular electrophysiology is critically dependent on the characterization of ion channel kinetics. This characterization is in turn critically dependent on mathematical models of the kinetics, but there is little consensus on which recordings to use (and how to use them) to derive the models. As a result, different literature models of the same current often provide very different predictions. We present a method for rapidly exploring and characterizing ion channel kinetics, using the hERG channel, responsible for cardiac IKr current, as an example. We fit a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp. The model is then used to predict over 5 minutes of recordings in the same cell in response to further voltage clamp protocols, including a new collection of physiological action potentials. Our technique allows rapid collection of data from single cells, produces more predictive ion current models than traditional approaches, and will be widely applicable to many electrophysiological systems.
Calcium induced Calcium Release (CICR) is a central mechanism on the pathway from electrical signal to contraction of a cardiomyocyte. It involves a wide range of spatial scales, from the nanometer wide diadic cleft spaces between junctions of the Sarcoplasmic Reticulum (SR) and the plasma membrane to cell wide depolarization, as well as a wide range of timescales from sub-millisecond channel state transitions to dynamics that emerge only over the course of many heartbeats. We present a model that encompasses all spatial and temporal scales of Calcium signaling in the left ventricular cardiomyocyte and their interconnections in high detail and precision. Cell wide reaction-diffusion equations describe the intracellular concentration fields of Calcium in the cytosol and SR. The source terms of these PDEs are (partially) generated by highly stochastic, very localized, strong Calcium currents that originate in the diadic clefts. The clefts contain representations of individual channels as discrete, continuous time Markov chains, coupled by local gradients inside the diadic space. Individual cleft spaces are coupled by the diffusion equations in the cytosol and SR, and the cell wide membrane potential, which is modelled by a set of ordinary differential equations (ODEs). The Monte-Carlo simulations of the channel dynamics lead to a high frequency of significant changes in the PDEs, which would limit the Finite Element PDE solver's time step size beyond practicallity. We apply a novel time step management and linear estimates of the PDE solution on short time scales to lift this restriction and render simulations efficient.
It is great fun to clarify or understand the physiological mechanism not only for scientists but also for students. So far, however, it has not been easy to study the dynamic properties of biological functions by reading textbooks. A large variety of simplified mathematical models have been developed in the field of Mathematical Physiology, and these are very useful to elucidate the principles of the biological functions. However, it is getting even more difficult to explain how the basic properties are build up by a large number of molecular details revealed in modern biophysical studies accompanied with positive and negative feedback loops. Our group is trying to rearrange the components of the comprehensive ventricular cell model into a learning system, e-Heart, including the basic function of ion channels, ion exchangers, simple cardiac action potentials, homeostasis of the ionic composition, cell volume, myofilament contraction, enzyme reactions, energy metabolism, and excitation propagation. The models should be user-friendly for physiologist as well as students to allow simple simulation experiments of modifying model equations to observe responses on the computer graphics with practical knowledge of computation.
We present recent developments on reproducibility, reusability, and discoverability of computational models in physiological systems. In doing so, we aim to improve the comprehensibility of models and associated data. Specifically, we will present the Physiome journal, an under-development Physiome web-portal, and provide some updates on various community standards that enable these efforts to proceed.
The Physiome journal was launched at the 38th World Congress of the IUPS, as an effort to encourage the reproducibility and reuse of models by providing citation credit for papers that describe and document curated and annotated models. We will introduce Physiome, and discuss the still evolving curation and annotation workflows, which are used to evaluate submissions to Physiome and the mechanisms by which we hope to track model reuse and measure impact.
Several recent advances in community standards and core infrastructural tools have been key in establishing Physiome. Advances to be covered include: those in OpenCOR and the Physiome Model Repository; the COMBINE Archive and the Open Modeling EXchange format; the harmonization of semantic annotations for computational models in biology; and recent work to make use of semantic annotations in a model discovery tool. This work is primarily based on the standards CellML and SED-ML, but other relevant standards will also be introduced.
Finally, a new effort has been launched to try and pull all these various efforts into a common, web-based, platform known as the "Physiome Portal". Preliminary work on this will be presented and future directions discussed.
Cardiac output, driven by metabolic demand, is the consequence of a cascade of electro-fluid-mechanical function spanning both spatial and temporal scales. Core to this function are myocardial energetics at the cellular level, driving the contractile cycle of the heart. At the organ-scale, the mechanics of the myocardium and hemodynamics rely on the efficient transmission of cellular work. However, as disease processes alter the normal mechanical function of the ventricle, this transmission can be significantly altered. Understanding the mechanical and hemodynamical influence in disease is then a critical step for under the work burden of the heart. In this talk, we talk about recent work examining the integration of biomechanical principles with magnetic resonance imaging (MRI) to understand the sources of mechanical loss at the whole-organ scale. The mechanical impact of hemodynamic losses is explored using phase contrast (MRI) and virtual work energy relative pressure relations, providing a noninvasive technique for probing flow energetics. Within tissue, we explore mechanical function and the impact of viscoelasticity using cardiac MR elastography.
The electrogenic Na/HCO3 cotransporter (NBCe1), by acting as “acid extruder” to increase
intracellular pH (pHi), regulates pHi in nearly every cell type in the body. In cardiac
myocytes, NBCe1 action is an important modifier of contractility and excitability, ischemic
and reperfusion injury, hypertrophy and heart failure.
Despite its name of “bicarbonate” transporter, experimental evidence from studies of
NBCe1 in brain slices and Xenopus oocytes show that inhibition of extracellular carbonic
anhydrase (CA) accentuates the magnitude of the extracellular acidification caused by
NBCe1-mediated acid extrusion. This phenomenon is consistent with the hypothesis that
the dominant, if not the only, substrate of NBCe1 is carbonate rather than bicarbonate.
In this study, we employ a reaction-diffusion mathematical model of a Xenopus oocyte co-
expressing NBCe1 and (extracellular) CAIV to study the surface pH (pHS) changes caused
by the influx of two bicarbonate ions versus the influx of one carbonate ion (“carbonate
model”) in the presence or absence of ACZ—a CA inhibitor. In agreement with
experimental data, the “carbonate model” predicts pHS changes that are markedly
accentuated by ACZ, leading us to conclude that NBCe1 is not a pure bicarbonate
By performing additional simulations with simultaneous influxes of carbonate and bicarbonate—having different carbonate/bicarbonate molar ratios but same total net flux— we conclude that carbonate is the dominant substrate of NBCe1.
Torsade de pointes is a potentially lethal ventricular arrhythmia and one of the most serious side effects of drugs. Recently, we proposed a novel proarrhythmic risk assessment system combining in vitro channel assays and in silico simulation of cardiac electrophysiology using a multi-scale simulation model of the human heart (UT-Heart) . We extend this approach and report a comprehensive hazard map of drug-induced arrhythmia based on the exhaustive in silico ECG database of drug effects, developed using the RIKEN K-computer computer with a performance of over 10 petaflops (SPARC64 VIIIfx, 705024 cores, Fujitsu Ltd. Kawasaki, Japan). We varied the inhibition rate of five ionic currents known to affect arrhythmogenicity, including IKr, INa, INaL, ICaL and IKs, to simulate a total of 9075 patterns of multiple ionic current inhibitions. For every combination of current inhibition, we simulated the electrophysiological activity of the heart and the associated 12-lead ECG to create a five-dimensional map of arrhythmia risk, in which the co-ordinates of the map represent the inhibition rate of each
current. This database will be freely available at http://ut-heart.com/; thus, it can be used not only to assess the risk of drug
candidates at any stage of R&D, but also to serve as a tool to design a safe drug without resorting to animal or clinical studies.
Okada, J. et al., Science Advances 1, (2015).
Okada, J. et al. Am J Physiol 301, H200-208 (2011).
Washio, T., Okada, J. & Hisada, T. SIAM Review 52, 717-743 (2010).
Cardiac resynchronization therapy (CRT) remains a successful treatment option for patients with dyssynchronous heart failure, even though up to 30% of the patients do not respond the treatment. Several previous studies have shown that heterogeneities in mechanical and electrical function in the dyssynchronous heart can be used as predictors of successful CRT outcome. For example, results from a recent computational modeling study in our group found that during acute CRT, the degree of reduction of septal myocardium performing negative work correlates with the degree of left ventricular reverse remodeling at 6 months of CRT. These work-based indicators of the likely success of the procedure are consistent with improvements in cardiac work after CRT.
To better understand mechanisms of the long-term remodeling responses to CRT, we have developed biomechanical computational growth models that can simulate heterogeneous remodeling responses due to long-term bi-ventricular pacing. Previous studies have shown regional work performed by the cardiomyocytes correlated with local tissue growth and likely play an important role in cellular responses underlying mechanisms of adverse and reverse remodeling. By implementing a work-based growth law into a finite element model of dyssynchrony and subsequent resynchronization, we have shown that acute redistribution of local myocardial work after CRT can be used to predict the amount of long term remodeling in both responders and non-responders. A validated, mechanistic multi-scale model of the long term remodeling post-CRT can be used to better understand cell-scale work and metabolic changes in CRT, and could be part of the decision support process to improve outcome of the procedure.
Under the CiPA (Comprehensive In Vitro Proarrhythmia assay) initiative led by FDA, a new paradigm for torsades de pointes (TdP) risk assessment has been proposed with emphasis on in-silico integration of drug-induced multi-channel block effects observed in in-vitro ion channel screening assays for improved TdP risk prediction. A large amount of work has been carried out in the field with development of several excellent TdP risk classifiers based on drug-induced multi-channel blockage data. These classifiers can be broadly categorized into two categories: 1] The classifiers built directly on in-vitro data itself (direct features), 2] The classifiers that are built on features extracted from output of drug-induced multi-channel blockage simulations in the in-silico models (derived features). The derived features obtained from complex biophysical models thus far have not provided consistent improvement in TdP risk assessment, and hence question the value of including complex biophysical details. Here, we examine the predictive capability of direct-feature based classifiers on several of the previously reported in-vitro datasets using a new two-step method. The proposed method allows to isolate the effects of hERG and non-hERG channels in the classification problem. For the several in-vitro assay datasets tested, our results show that the direct-feature based classifiers developed using the proposed approach provides comparable or superior risk prediction to existing classifiers built on derived features extracted from complex biophysical models. We also highlight the link between the direct and derived feature based classifiers.
Background: The high activation rates during atrial fibrillation (AF) increase oxygen demand. We have shown that acute AF causes supply/demand ischemia.
Objective: Characterize early AF-induced remodeling of atrial mitochondrial function after one week of induction.
Methods: AF was mimicked in Landrace pigs by one week of rapid atrial pacing (RAP). Subsequently, pigs were anesthetized and the chest was opened to freeze and collect fresh tissue to perform structural, biochemical and functional studies.
Results: After one week of RAP, the energetic status was altered as evidenced by a decrease in phosphocreatine to inorganic phosphate ratio when compared to control. This was associated with a reduction in mitochondrial size, a decrease of cardiolipin (CL) content, and an increased oxidized CL to CL ratio. Mitochondrial hydrogen peroxide production was decreased secondary to a twofold increase in its total peroxidase activity. RAP decreased mitochondrial basal oxygen consumption in presence of fatty acid, while their ADP-stimulated respiration was increased in presence of glutamate+malate (GM). During fatty acid oxidation, RAP induced 1) depolarization of mitochondrial membrane potential under phosphorylating conditions and 2) increase the sensitivity of the mitochondrial permeability transition pore to calcium in presence of cyclosporin A. RAP mitochondria showed an increase in their rate of calcium uptake in presence of GM. RAP atria showed glucose-6-phosphate accumulation and increased activity of bound mitochondrial hexokinase.
Conclusion: RAP causes early remodeling of atrial mitochondrial function and structure. Although mitochondrial function is largely preserved, the energy production capacity seems insufficient to meet demand, possibly contributing to AF progression.
Background: Calcium “leak” from the sarcoplasmic reticulum (SR) caused by RyR2 dysfunction can promote delayed after depolarization and arrhythmia.
Objective: To test whether the SR calcium “leak” can influence ventricular arrhythmia susceptibility and/or maintenance by a mechanism involving the mitochondrial permeability transition pore (mPTP).
Methods: Burst pacing sequences were used to induce sustained or non-sustained ventricular arrhythmia in the perfused rat heart. Pseudo electrocardiogram (p-ECG), left ventricular pressure, oxygen consumption, mitochondrial membrane potential and intracellular calcium were monitored. p-ECG signals were used to assess arrhythmia duration and complexity. MPTP activity was assessed by the calcein entrapment method. Hearts were perfused with tacrolimus (FK506) alone, or in combination with cyclosporin A (CsA) to promote SR calcium “leak” or inhibit mPTP, respectively.
Results: FK506 perfusion promoted a CsA-sensitive mitochondrial depolarization. In presence of FK506 mPTP was activated (i.e. mitochondrial membrane depolarization) following a short burst-pacing sequence. Susceptibility to arrhythmia was increased following mPTP activation. Long burst-pacing sequence in the presence of FK506 induced sustained (> 300 s) ventricular arrhythmias, while co-perfusion with CsA lead to spontaneous defibrillation in less than 9 s. This defibrillating effect of CsA was associated to less mitochondrial membrane potential depolarization and a decrease in intracellular calcium overload induced by FK506. The increased arrhythmogenicity induced by FK506 was counteracted by Glibenclamide an inhibitor of the ATP-dependent potassium channel (IKATP).
Conclusions: SR calcium leak associated to an external stress leads to mPTP activation. Our data suggest that the consequent energetic depression opens IKATP thus favoring ventricular arrhythmia maintenance.
Since the launch of the CiPA initiative, human-based computer models of cardiac electrophysiology have become increasingly popular in safety pharmacology, for early prediction of drug-induced arrhythmias. However, the high variability in drug responses makes predictions for the whole population based on a single computer model challenging. In addition, arrhythmias are likely to occur in diseased patients, already having some cardiac disorders or taking concomitant medications. The aim of this study is to investigate how computer simulations in populations of human diseased action potential (AP) models can predict clinical risk of Torsade de Pointes (TdP). An in silico population of human ventricular cells was constructed based on the O'Hara-Rudy model and human experimental data. Ionic current distributions in the population were chosen to represent electrophysiological changes recurrent in cardiovascular diseases, e.g. reduced Na+-K+ pump activity, and increased Ca2+ or Na+-Ca2+ exchanger currents. Multichannel drug action was simulated for 40 reference compounds using IC50 values and concentrations up to 100-fold the maximal effective free therapeutic concentration. Drug-induced changes in AP were quantified, together with occurrence of repolarisation abnormalities. TdP risk was defined based on CredibleMeds. Repolarisation abnormalities occurrence in silico predicts clinical TdP risk with 90% accuracy (92% specificity, 89% sensitivity). All drugs with high TdP risk were correctly identified. Only 3 drugs with potential/conditional TdP risk were misclassified as safe (Amitriptyline, Ivabradine, Nicardipine) and 1 safe drug (Mexiletine) as risky. Human in silico drug trials suggest RA occurrence in diseased populations as an effective biomarker to predict clinical TdP risk.
Computational cardiac electrophysiology models that are capable to deliver physiologically reliable 3D ventricular activation maps and the corresponding body surface ECGs are currently only feasible on supercomputers. This fact severely limits the applicability of such models in the clinical practice. Modern ECG imaging tools rely on simplified electrophysiology models, that do not account for the heterogeneity and anisotropy in the electric conductivity of the heart and the torso. Additionally, these tools are often limited to the epicardium only, providing no insights on the 3D volumetric activation.
In this talk we present a software based on the eikonal model and the lead-field approach, that simulates the 3D ventricular activation at 1mm resolution and the 12-lead ECG at 1kHz sampling rate almost realtime on modern GPGPU architecture. For 6 patient-tailored anatomies, we validated this new simple model against bidomain simulations performed on a supercomputer with respect to accuracy in the ventricular activation and ECG. For all 6 patients, the ventricular activation compares well between our model and the bidomain. Corresponding QRS-complexes, are also accurately reproduced in terms of duration, amplitude, shape and signed area. Calculations are performed within 0.3 seconds (best case) to 0.8 seconds (worst case). The eikonal model with the lead-field approach can provide patient-tailored, ventricular activation and surface ECG almost real-time on a laptop or desktop computer. This makes the method suitable for ECG imaging techniques and interactive simulation tools, bridging the gap between the clinical practice and state-of-the-art cardiac electrophysiology modelling.
Tricyclic antidepressants are leading cause of mortality from suicidal intoxications. One of their lethal mechanisms is
cardiotoxicity expressed by cardiac channel blocking and anticholinergic tachycardia . The aim of the study was to simulate
amitriptyline overdose effect on human electrophysiology.
Amitriptyline and its metabolite nortriptyline time-concentrations profiles in plasma and cardiac tissue were simulated in a PBPK model with permeability-limited heart model nested in. The models accounting for inter-individual variability were written in R v.3.4.0.
Ten iterations of simulation scenario following case study of 34-year-old male intoxicated with 750 mg of amitripty line were run . Predicted individual free cardiac concentrations were combined with patient-specific parameters, and in vitro channel inhibition to simulate pseudoECG traces in Cardiac Safety Simulator (CSS)  and ΔQTcB as an endpoint. The Emax model describing relation between plasma amitripty line concentration and RR was fitted to clinically observed data. The parameters of Emax model were as follows: E0 [ms]- 995.3; Emax [ms] - 500.8; EC50 [μM]- 0.4; n -1.5. Simulated ΔQTcB were in the range 415 - 501 ms, with mean: 460 +/- 34 ms compared to clinically observed value of 488 ms. The simulated tachycardia and QTc prolongation related to amitriptyline poisoning were in accordance with in vivo toxicological observations. The results of this study support the predictive abilities of PBPK/PD modelling and simulations.  Minges P.G., Shaffer R.W. (2017) Hyzy R. (eds) Evidence-Based Critical Care. Springer, Cham  Paksu S., et al. (2014) Hum Exp Toxicol 33(9):980-990.  Glinka A., Polak S. (2015) Toxicol Mech Methods 25(4):279-286.
Optical mapping (involving the use of fluorophores sensitive to specific parameters, such as voltage or calcium) is an established non-contact technique that has been essential in advancing our understanding of cardiac electrophysiology and arrhythmogenesis. As with any technique, however, it is important to consider factors that may influence its results. Here I will highlight some of the metabolic, mechanical, and electrical considerations relevant for optical mapping that have come out of our experience with studies of ischemia and infarction.
Ischemia is characterised by metabolically-driven heterogeneity in electrophysiological and mechanical function. With optical mapping, electrophysiological heterogeneity will lead to light penetration-dependent signal distortion, while the influence of mechanical heterogeneity on electrical activity will be eliminated by the required uncoupling or constraint of contraction. In the isolated rabbit heart, we have visualised ischemia-induced transmural electrophysiological heterogeneity with dual wavelength excitation, and have demonstrated a calcium-mediated mechanical contribution to arrhythmogenesis by modulating contraction. In infarction, structurally-based heterogeneities also exist, including altered cellular composition of cardiac tissue. Cell-specific contributions to electrical activity, however, are lost with the use of indiscriminate fluorescent dyes. By the cell-specific expression of a voltage-sensitive fluorescent protein in mice, we have recorded action potential-like signals in cardiac non-myocytes at the scar border of cryoinjured hearts, providing direct evidence of heterocellular electrotonic coupling in native myocardium.
Overall, our experience with studies of ischemia and infarction illuminates metabolic, mechanical, and electrical factors that should be considered in optical mapping studies of cardiac electrophysiology and suggests the need to rethink traditional approaches to the use of this transformative technology.
Recent advances in human pluripotent stem cell (hPSC) biology enable derivation of essentially any cell type in the human body, and development of three-dimensional (3D) tissue models for drug discovery, safety testing, disease modelling and regenerative medicine applications. However, limitations related to cell maturation, vascularization, cellular fidelity and inter-organ communication still remain. Relying on an engineering approach, microfluidics and microfabrication techniques our laboratory has developed new technologies aimed at overcoming them.
Since native heart tissue is unable to regenerate after injury, induced pluripotent stem cells (iPSC) represent a promising source for human cardiomyocytes. Here, biological wire (Biowire) technology will be described, developed to specifically enhance maturation levels of hPSC based cardiac tissues, by controlling tissue geometry and electrical field stimulation regime (Nunes et al Nature Methods 2013). We will describe new applications of the Biowire technology in engineering a specifically atrial and specifically ventricular cardiac tissues, safety testing of small molecule kinase inhibitors, potential new cancer drugs, and modelling of left ventricular hypertrophy using patient derived cells.
For probing of more complex physiological questions, dependent on the flow of culture media or blood, incorporation of vasculature is required, most commonly performed in organ-on-a-chip devices. Current organ-on-a-chip devices are limited by the presence of non-physiological materials such as glass and drug-absorbing PDMS as well as the necessity for specialized equipment such as vacuum lines and fluid pumps that inherently limit their throughput. An overview of two new technologies, AngioChip (Zhang et al Nature Materials 2016) and inVADE (Lai et al Advanced Functional Materials 2017) will be presented, that overcome the noted limitations and enable engineering of vascularized liver, vascularized heart tissues and studies of cancer metastasis. These platforms enable facile operation and imaging in a set-up resembling a 96-well plate. Using polymer engineering, we were able to marry two seemingly opposing criteria in these platforms, permeability and mechanical stability, to engineer vasculature suitable for biological discovery and direct surgical anastomosis to the host vasculature.
Finally, to enable minimally invasive delivery of engineered tissues into the body, a new shape-memory scaffold was developed that enables delivery of fully functional tissues on the heart, liver and aorta through a keyhole surgery (Montgomery et al Nature Materials 2017).
Assessment of HERG/IKr block is crucial for pre-clinical drug screening. We have previously shown that morpholino anti-sense (AS) oligonucleotides increases functional HERG expression in HEK293 cells (Gong et al, 2014) by inhibiting the formation of a non-functional HERG isoform. Here, we determined if morpholino oligonucleotides increase IKr expression in human pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Following a 48 hr treatment with morpholino oligonucleotides, we observed an increase in IKr density (Vehicle: 0.18+0.03 (n=18), Morpholino: 0.33+0.06 (n=22), p<0.05). The increase in current density coincided with a shift in the distribution of cells expressing larger IKr current densities in the morpholino AS oligonucleotide-treated cells. Following morpholino oligonucleotide treatment, the percentage of cells expressing current densities >0.2 pA/pF increased from 39% to 68%. In addition, morpholino treatment did not change the HERG current properties. The data demonstrate for first time that morpholino AS oligonucleotide treatment increases IKr expression in hiPSC-CMs, resulting in a higher frequency of cells expressing larger IKr current densities. An increased IKr expression in hiPSC-CMs will allow for more accurate screening of IKr-induced drug effects on action potentials in both manual and multi-planar patch electrophysiology systems.
The extrinsic modulation of cardiac energy substrate metabolism is exerted by neural mediators and peptide hormones. Most studies on hormonal regulators have been focused on insulin and glucagon-like peptide, which are particularly interesting also for their therapeutic use. However, there is a vast array of other peptide or protein hormones involved in the systemic homeostasis of energy substrate metabolism, which might also exert a direct action on cardiac energy turnover. To date, this aspect of cardiac physiology and pathophysiology has been little explored. The gut-derived hormone ghrelin, especially its acylated form, plays a major role in the regulation of systemic metabolism and exerts also relevant cardioprotective effects. We provided direct evidence, in vivo, that acute increases in des-acyl ghrelin or acyl ghrelin do not interfere with cardiac metabolism in normal heart, while they enhance free fatty acid oxidation and reduce glucose oxidation in the failing heart, thus partially correcting its metabolic alterations. Another circulating factor that we are currently studying is follistatin-like protein 1 (FSTL1), one of the “myokines/cardiokines”, i.e. peptides and proteins secreted by skeletal muscle fibers and cardiomyocytes. Our recent data indicate that chronic FSTL1 infusion stably normalizes cardiac free fatty acids, glucose, ketones consumption and systemic respiratory quotient and moderately improves diastolic and contractile function in heart failure. Ghrelin and FSTL1 are interesting also because they can be synthesized by the heart, hence they might contribute to the mutual endocrine regulation of cardiac and systemic energy metabolism.
Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. The rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy will be summarized. The approach is to upregulate ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.
Fibroblasts constitute a significant cell population in the myocardium of the normal and diseased heart. Their primary physiological function is synthesis of extracellular matrix proteins. Various stimuli can trigger differentiation of fibroblasts into myofibroblasts, which are a major contributor to myocardial remodeling in heart disease. Despite extensive research efforts the physiological and pathophysiological roles of cardiac fibroblasts remain ill-defined. In this presentation, we will provide an overview of studies from us and others on mathematical modeling and microscopic imaging of fibroblasts in cardiac tissues. We will describe mathematical models that allow us to explore potential contributions of fibroblasts to cardiac conduction. In particular, we introduce an approach for multidomain mathematical modeling of tissue electrophysiology (Sachse et al, Ann Biomed Eng. 2009;37(5):874-89), which considers myocardium as a mixture of myocytes and fibroblasts with varying electrophysiological properties and intercellular electrical coupling. Also, we will provide insights into our attempts to establish a microstructural basis for multidomain and other types of conduction modeling (Seidel et al, Ann Biomed Eng. 2016; 44(5):1436-1448). The approach is based on immunolabeling, high-resolution three-dimensional confocal microscopy and image quantification. Finally, the presentation will shed light on consequences of fibroblast activation in the diseased heart, specifically, fibrosis. We will discuss potential fibrosis-related effects on conduction, excitation-contraction coupling and metabolism.
Introduction: After infarction compensated remodeling of the left ventricular (LV) may be followed by adverse remodeling leading to heart failure. The mechanism of adverse remodeling maybe linked to the elevated wall stress in the dysfunctional myocardium adjacent to the infarct (border zone-BZ). We hypothesize that increased BZ stress results in altered metabolism which could drive the transition from compensated to adverse remodeling. To evaluate BZ and remote metabolism we compared the regional uptake and intracellular conversion of 1-13C-pyruvate using hyperpolarized (HP) 13C MR.
Methods: An established pre-clinical posterolateral infarct model of LV remodeling was used to investigate region metabolism. To accurately measure regional metabolism, we developed implantable carbon-tuned surface coils placed on the epicardial surface over the BZ and remote regions. A coronary catheter was placed for direct injection of the HP substrate to maximize deliver and eliminate cavity blood pool signal. MR was performed at 6-weeks post infarct with a spectra acquired every 1.5s for each region simultaneously during HP infusion under physiologic and DOB stress conditions. The resulting spectra from each coil were analyzed to measure lactate, alanine, bicarb, and total flux.
Results: Under physiologic (Pre-DOB) conditions the percent difference between remote and BZ lactate, alanine, and total flux was only slightly elevated in the remote region whereas bicarb flux was greater in BZ compared to remote (Fig). DOB stress produced an increase in remote metabolite flux compared to BZ with lactate, alanine and total flux reaching significance and bicarb flux shifting from greater in BZ Pre-DOB to greater in remote.
Conclusion: These findings demonstrate an impaired metabolic response to pharmacologic stress in BZ myocardium which may provide a mechanism for the established association of mechanical stress and adverse cardiac remodeling following infarct.
An undesirable side effect of drugs are cardiac arrhythmias. The current assessment of drug toxicity involves costly tests that hinder efficient drug development. In this work, we establish a high fidelity multiscale computational model of cardiac electrophysiology to predict the onset of Torsades de Points in the presence of drugs. Using this model, we create a proarrhythmic phase diagram and identify safe and arrhythmic regions for varying degrees of hERG (Kr) channel and Cav1.2 (CaL) channel block. We demonstrate that—provided we know the drug concentration required to achieve a 50% block of these two channels—our phase diagram can correctly classify the risk categories of 27 out of 31 common drugs. We also show that the QT interval alone displays poor specificity to stratify risk categories: For similar probabilities of Torsades de Pointes, the QT interval can vary from 410 to 500 ms. Our study suggests a novel and efficient strategy to estimate the risk of new compounds and highlights the role of the Cav1.2 channel as an inhibitor of Torsades de Pointes.
Drugs often have undesired side effects. In the heart, they can induce lethal arrhythmias such as Torsades de Points. The risk evaluation of a new compound is costly and can take a long time, which often hinders the development of new drugs. Here we establish an ultra high resolution, multiscale computational model to quickly and reliably assess the cardiac toxicity of new and existing drugs. The input of the model is the drug-specific current block from single cell electrophysiology; the output is the spatio-temporal activation profile and the associated electrocardiogram. We demonstrate the potential of our model for a low risk drug, Ranolazine, and a high risk drug, Quinidine: For Ranolazine, our model predicts a prolonged QT interval of 19.4% compared to baseline and a regular sinus rhythm at 60.15 beats per minute. For Quinidine, our model predicts a prolonged QT interval of 78.4% and a spontaneous development of Torsades de Points both in the activation profile and in the electrocardiogram. We also study the dose-response relation of a class III antiarrhythmic drug, Dofetilide: At low concentrations, our model predicts a prolonged QT interval and a regular sinus rhythm; at high concentrations, our model predicts the spontaneous development of arrhythmias. Our multiscale computational model reveals the mechanisms by which electrophysiological abnormalities propagate across the spatio-temporal scales, from specific channel blockage, via altered single cell action potentials and prolonged QT intervals, to the spontaneous emergence of ventricular tachycardia in the form of Torsades de Points. We envision that our model will help researchers, regulatory agencies, and pharmaceutical companies to rationalize safe drug development and reduce the time-to-market of new drugs.
Following myocardial infarction (MI), either insufficient or excessive wound healing can detrimentally affect cardiac remodeling. During the inflammatory phase fibroblasts adopt a degradative phenotype, while in the proliferative phase they drive fibrosis. Attempts to therapeutically control fibroblast-mediated matrix remodeling are substantially hindered by the complexity of their extracellular and intracellular signaling networks. To address this challenge, we have developed a logic-based model of the cardiac fibroblast signaling network, validated against extensive independent literature and our own experiments. The model predicts distinct molecular drivers of fibroblast phenotypes in contexts representative of inflammatory or proliferative post-MI environments. Integration of this model with drug-target databases enabled virtual drug screening for FDA-approved drugs that are predicted to specifically modulate fibroblast activity in inflammatory or proliferative phases.
Since oxidative phosphorylation in mitochondria utilizes proton gradient across the inner mitochondrial membrane, cytosolic pH is supposed to influence mitochondrial functions. The in situ influences are, however, not well understood due to difficulties in wet experimental measurements. In this study, we investigated effects of respiratory acidosis on myocardial mitochondrial functions by means of computer simulation. Saito et al (J Physiol, 2016) published a detailed mathematical model of myocardial mitochondria including electron transport chain, ATP synthase, Krebs cycle, and membrane transporters. We integrated the mitochondria model into a comprehensive cell model (Matsuoka et al, unpublished), which formulates membrane excitation, pH regulation, and ATP- and pH-dependent ion transport and contraction. The integrated model successfully reproduced increases in intracellular Na, K, Ca, and bicarbonate under respiratory acidosis by raising extracellular CO2 pressure.
Simulation results under acidotic conditions showed growth of both cytosolic ATP consumption, which is attributable to Na-K pump, and mitochondrial ATP production. ATP synthase was activated under the acidotic conditions despite decreases in pH gradient. This activation is attributed to elevated mitochondrial inner membrane potential, which is generated by electron transport chain. All of Complex I, III, and IV were up-regulated by increased NADH, and NADH-forming reactions by ICDH, MDH and PDHC were promoted because of increases in their substrates. These computational predictions will provide working hypotheses for experimental studies.
The ability to create in vitro 3D cardiac tissue that closely recapitulates the electromechanical properties of native myocardium is crucial to the study of cardiac diseases, pharmacological testing, and the qualification of cells for tissue-engineered regenerative medicine.
A variety of approaches have been implemented to engineer 3D cardiac constructs, including different scaffolds and cells types, and to interrogate the active and passive mechanical and electrical properties; however, they were designed to measure either isotonic contraction or auxotonic contraction, without the ability to control the applied tension.
Here we demonstrate the I-Wire platform, which aims to overcome the aforementioned drawbacks. The I-Wire comprises a six-well plate with an inserted PDMS casting mold to grow 3D cardiac tissue constructs and a microscope-based imaging system for optical registration of contraction and detection of [Ca2+]i and Vm. This platform allows controlling the force applied to the engineered cardiac tissue constructs (ECTCs) and examining their passive and active mechanical properties.
The system was tested using rat neonatal cardiac cells and hiPSC-derived cardiomyocytes. The ECTCs recapitulated the Frank-Starling force-tension relationship, and demonstrated expected transmembrane action potentials, electrical and mechanical restitutions, and response to β-adrenergic stimulation. The optical registration of the flexible sensor movement allows quantitative measuring of contraction forces under different auxotonic loading conditions and pharmacological interventions. The quantitative measurements and modeling enabled by our I-Wire platform have great potential in pharmacology, cardiotoxicity, and basic science to investigate mechanisms of cardiac disease in both transgenic animal models and in human 3D cardiac tissue.
Despite extensive studies attempted to identify clinical indices for the selection of patient candidates who benefit from cardiac resynchronization therapy (CRT), there still remains a significant number of non-responders among those treated by this invasive therapy. A multi-scale heart simulation capable of reproducing the electrophysiology and mechanics of a beating heart may help resolve this problem1. In a retrospective study including nine patients, we tested whether we can reproduce the response to CRT using the patient-specific multi-scale heart simulator2. Based on the clinical data collected before CRT, we created patient-specific failing heart models with conduction block. Each model was tailored to reproduce the surface electrocardiogram and hemodynamics of each patient. To each heart model, we performed bi-ventricular pacing according to the actual pacing protocol and compared the results with the clinical data recorded after the treatment. CRT simulation improved the electromechanical dyssynchrony by varying degree for each patient. The best correlation between simulated and clinical results was obtained for the simulated maximum value of the time derivative of ventricular pressure (dP/dtmax) and the clinically observed improvement in the ejection fraction (EF). By integrating the complex pathophysiology of the failing heart with conduction block, patient-specific, multi-scale heart simulation could successfully reproduce the response to CRT. With further verification and improvement through the prospective study, this technique could be a useful tool in clinical decision making.
1 Panthee, N. et al Med Image Anal (2016). 2 Okada, J. et al., J Mol Cell Cardiol (2017).
The intermediary metabolism of energy providing substrates is the best characterized, dynamic biological network to date. In a nutshell, cardiac metabolism serves the following major functions: 1. Energy transfer. 2. Redox regulation. 3. Acid/base regulation. 4. Signaling. 5. Provision of biosynthetic materials. 6. Protein, RNA, and DNA modifications.
Decoding and comparing metabolic phenotypes by flux balance analysis and other contemporary analytical techniques is a major challenge ahead. New leads for cardiac metabolism arise from the burgeoning literature on cancer cell metabolism. "I never thought I'd ever have to learn the Krebs cycle. Now I realize I have to." (James Watson, The New York Times, May 15, 2016).
In experimental studies on cardiac tissue, the end-systolic force-length relation (ESFLR) has been shown to depend on the mode of contraction: isometric or isotonic. The isometric ESFLR is derived from isometric contractions spanning a range of muscle lengths while the isotonic ESFLR is derived from shortening contractions across a range of afterloads. The ESFLR of isotonic contractions consistently lies below, and to the right of, its isometric counterpart. Despite the passing of over a hundred years since the first insight by Otto Frank, the mechanism(s) underlying this protocol-dependent difference in the ESFLR remain incompletely explained. Previous experimental studies have compared the dynamics of isotonic contractions to those of a single isometric contraction at a length (Lo) that produces maximum force, without considering isometric contractions at shorter muscle lengths. Here we used a mathematical model of cardiac excitation-contraction to simulate isometric and work-loop contractions, and compared their Ca2+ transients under equivalent force conditions. We found that the duration of the simulated Ca2+ transient increased with decreasing sarcomere length for isometric contractions, and increased with decreasing afterload for work-loop contractions. At any given force, the Ca2+ transient for an isometric contraction was wider than that of a work-loop contraction. By driving work-loops with Ca2+ transients of greater duration, we found that the duration of muscle shortening was prolonged, thereby shifting the work-loop ESFLR towards the isometric ESFLR. These observations are explained by the mechanism of force-dependent affinity of Ca2+ binding, whereby an increase of force increases the rate of binding of Ca2+ to troponin-C.
Frequency-dependences of action potential and Ca2+ transients are fundamental features of cardiac ventricular myocytes and should be considered in mathematical models. We studied the frequency-dependences in isolated mice ventricular myocytes and investigated roles of mitochondrial Na+/Ca2+ exchange (NCLX) on action potential and Ca2+ transient. Action potentials were measured by patch clamp method and Ca2+ transients were measured by using Ca2+-sensitive dye (Cal-520, AM) and EM-CCD camera (ImagEM, Hamamatsu Photonics). In amphotericin B perforated patches, action potential duration (APD) of 90% repolarization (APD90) was significantly shortened as stimulus frequency was changed from 0.5 to 10 Hz, but APD of 30% repolarization (APD30) tended to increase. Field stimulation of Cal-520, AM-loaded myocytes demonstrated shortening of Ca2+ transient duration (CaD) at 90% recovery (CaD90) with increasing stimulus frequency. The roles of NCLX on action potential and Ca2+ transients were studied in whole-cell patches by applying a blocker of NCLX (CGP-37157, 2 µM) from glass pipettes. CGP-37157 tended to shorten CaD90, suggesting a role of NCLX on SR Ca2+ uptake. This study provides basic features of the frequency-dependences of mice ventricular myocyte, which are useful in mathematically modelling action potential and Ca2+ transient of mice ventricular myocytes.
Intense physical exertion requires large ATP consumption and generates much heat. Maintaining energy homeostasis is a big challenge. Peaks of ATP consumption during skeletal muscle contraction and cardiac systole must be buffered. Muscle cells contain creatine kinase which can resynthesize ATP quickly. Computational model analysis of muscle energetics, integrating data on creatine kinase activity and the measured activation time of oxidative phosphorylation, reveals that intracellular transport of energy in the form of ATP is quite efficient. The creatine kinase system buffers ATP levels effectively, in particular preventing large swings in ADP concentration during the cardiac cycle or cyclic skeletal muscle contraction. This does not explain why the system contains distinct cytosolic and mitochondrial creatine kinase isoforms, but a possible function of these isoforms is suggested by computational model simulations. Uncertainty in the input data is taken care of by ‘sloppy modeling’ methods which use Markov chain Monte Carlo algorithms.
Efficient dynamic buffering of ATP at the cellular level would be meaningless if heat, threatening to ‘boil the tissue like an egg’, were not removed effectively from the large muscle mass. A whole body model of heat transport and temperature regulation indicates that pro cyclists’ bodies can get rid of 1600 Watt heat to the environment during the most challenging episodes in the Tour de France. Even on exceptionally steep slopes brain temperature does not get dangerously high. Paradoxically, brain temperature potentially might get higher when a pro cyclist climbs a high mountain pass in freezing conditions than under mild weather conditions.
In this study, in silico populations of atrial cells were built to investigate the effects of drugs on markers of arrhythmia. The effect of two drugs routinely used to treat atrial fibrillation (Flecainide and Dofetilide) were incorporated into simulations to investigate their effects on AP morphology and APD restitution properties at three different concentrations. The effects of ion channel block were modeled by tuning the maximal conductance of seven ionic currents: INafast, INalate, ICaL, IKr, IKs, IK1, and Ito, to match known properties of the drugs.
A population of cells in normal sinus rhythm was calibrated based on experimental data from literature. This guaranteed that all models tested presented normal AP morphologies before the drug effects were applied and thus exclude any model that showed unphysiological AP and calcium transient profiles. Biomarkers related to AP traces and Ca2+ transients were extracted at 1 Hz pacing. Furthermore, APD restitution curves were simulated under dynamic pacing. We observed repolarization abnormalities with both Flecainide and Dofetilide, including repolarization failure and afterdepolarizations. Populations exposed to high drug concentrations of ten times Cmax showed an increase in predisposition for APD alternans, with alternans occurrence as high as 71% with Flecainide at 3 times Cmax and 77% with Dofetilide at 10 time Cmax. Sensitivity analysis showed that observed AP, CaT and alternans biomarkers were governed by similar parameters, with sensitivities being exacerbated at high drug concentrations.
This study showcases the potential for the use of in silico trials in the assessment of drug induced clinical arrhythmias.
Atrial fibrillation (AF) is the most common cardiac arrhythmia. One of the pathological processes underlying AF is increased fibrosis. It has been shown that fibroblasts can couple electrotonically to cardiomyocytes. In this computational study, we investigate the effect of such coupling on electrophysiological properties of atrial cardiomyocytes.
We used 10 variants of a human atrial myocyte model, each parameterized to represent cells from different regions. All were studied when coupled to varying numbers of fibroblasts with varying coupling conductivities.
On the single cell level, myocytes from the pulmonary vein (PV) area were most affected: For instance, with two fibroblasts and a coupling conductivity of 2nS each, action potential amplitude decreased from 105mV to 94mV and upstroke velocity from 192V/s to 152V/s. The resting membrane voltage was elevated from -78.4mV to -72.8mV.
To assess possible pathological impact, we recorded restitution curves of the conduction velocity (CV), effective refractory period (ERP). CV restitution again showed the strongest impact of fibroblast coupling at the PV area, where CV deteriorated significantly at a basic cycle length (BCL) of 600ms (300ms without fibroblasts). ERP was significantly elevated in the PV area and valve rings for all BCLs, but slightly reduced in most other regions.
The PV area is of special interest in AF since it is often the source of triggers for irregular activity. We show that, additionally, the electrophysiology of PV myocytes is affected more than other regions by coupled fibroblasts. This may contribute to their crucial role in AF.
Long QT syndrome (LQTS) is a rare congenital and inherited or acquired heart condition in which delayed repolarization of the heart following a heartbeat increases the risk of episodes of torsades de pointes. In genetic analysis of R/O LQTS patient with arrhythmias during fetus period, we identified a novel SCN5A variant (A1656D) in a highly conserved region of the S4-S5 Intracellular Loop in Domain IV. We investigated whether this SCN5A missense mutation could form the genetic basis for LQTS in this patient. Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na+ channels revealed a defective inactivation in A1656D channel, consistent with LQT phenotype. In addition, we found that the subtle differences of Na+ channel blockers, ranolazine and mexiletine in correcting A1656D mutant channel activity. Incorporation of the A1656D mutation-induced biophysical changes in channel gating described above into a model of an adult human ventricular myocyte yielded simulated action potentials with markedly delayed repolarization that are qualitatively similar to the patient’s phenotype. Simulations suggest mexilentine as beneficial in recovering channel function consistent with the successful arrhythmia management obtained by mexiletine. The A1656D SCN5A mutation confers gain-of-function effects on Na+ channel activity. Reduction of a mutation-induced current by mexiletine suggests a therapeutic mechanism.
Sodium-glucose cotransporter 2 inhibitors (SGLT2i‘s; EMPA, CANA and DAPA)), targeting SGLT2 in the kidney (not present in heart), are the first diabetes drugs to successfully reduce hospitalization for heart failure in type 2 diabetes patients. However, how this kidney-targeted agent reduce cardiovascular diseases is as yet unknown. Elevated cardiac cytoplasmatic sodium and calcium concentration ([Na+]c, [Ca2+]c) and decreased mitochondrial calcium ([Ca2+]m) are drivers of heart failure and cardiac death. We therefore hypothesized that SGLT2i‘s directly modifies [Na+]c, [Ca2+]c and [Ca2+]m in cardiomyocytes. [Na+]c, [Ca2+]c, [Ca 2+]m and sodium-hydrogen exchanger (NHE)-activity were measured in isolated ventricular myocytes from rabbit, rat and mouse with the appropriate fluorescent probes. EMPA(gliflozin) treatment directly inhibited the NHE-flux, caused a reduction of [Na+]c and [Ca2+]c, and increased [Ca2+]m. After pre-incubation with the NHE inhibitor Cariporide, the effect of EMPA was strongly reduced. Similar effect were observed for CANA and DAPA. In isolated mouse hearts SGLT2i‘s induced vasodilation, and EMPA increased cardiac oxygen consumption. The data suggest that this new class of diabetic agents protects against heart failure and cardiac death through a direct effect on the heart. The [Na+]c-lowering class effect of SGLT2i‘2 is a potential approach to combat elevated [Na+]c that is known to occur in heart failure and diabetes.