Lufang Zhou

From mitochondrial ion channels to arrhythmias in the heart: computational techniques to bridge the spatio-temporal scales

Computer simulations of electrical behaviour in the whole ventricles have become commonplace during the last few years. The goals of this article are (i) to review the techniques that are currently employed to model cardiac electrical activity in the heart, discussing the strengths and weaknesses of the various approaches, and (ii) to implement a novel modelling approach, based on physiological reasoning, that lifts some of the restrictions imposed by current state-of-the-art ionic models. To illustrate the latter approach, the present study uses a recently developed ionic model of the ventricular myocyte that incorporates an excitation–contraction coupling and mitochondrial energetics model. A paradigm to bridge the vastly disparate spatial and temporal scales, from subcellular processes to the entire organ, and from sub-microseconds to minutes, is presented. Achieving sufficient computational efficiency is the key to success in the quest to develop multiscale realistic models that are expected to lead to better understanding of the mechanisms of arrhythmia induction following failure at the organelle level, and ultimately to the development of novel therapeutic applications.

A Reaction-Diffusion Model of ROS-Induced ROS Release in a Mitochondrial Network

Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS). Here, we develop a mathematical model of ROS-induced ROS release (RIRR) based on reaction-diffusion (RD-RIRR) in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and Ca2+ handling. Local mitochondrial interaction is mediated by superoxide (O2 .−) diffusion and the O2 .−-dependent activation of an inner membrane anion channel (IMAC). In a 2D network composed of 500 mitochondria, model simulations reveal ΔΨm depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O2.− diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that ΔΨm depolarization is mediated specifically by O2 .−. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell, thus it has important implications for understanding cell death during the progression of heart disease.

FROM MITOCHONDRIAL DYNAMICS TO ARRHYTHMIAS

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Delta psi(m), visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Delta psi(m), NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Delta psi(m) under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.

Modeling Cardiac Action Potential Shortening Driven by Oxidative Stress-Induced Mitochondrial Oscillations in Guinea Pig Cardiomyocytes

Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (DeltaPsi(m)) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K(ATP)) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K(ATP) current (I(K,ATP)). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of DeltaPsi(m), redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I(K,ATP), consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca(2+) handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.

Regulation of lactate production at the onset of ischaemia is independent of mitochondrial NADH/NAD+: insights from in silico studies.

Ischaemia decreases mitochondrial NADH oxidation, activates glycolysis, increases the NADH/NAD+ ratio, and causes lactate production. The mechanisms that regulate anaerobic glycolysis and the NADH/NAD+ ratio during ischaemia are unclear. Although continuous measurements of metabolic fluxes and NADH/NAD+ in cytosol and mitochondria are not possible in vivo with current experimental techniques, computational models can be used to predict these variables by simulations with in silico experiments. Such predictions were obtained using a mathematical model of cellular metabolism in perfused myocardium. This model, which distinguishes cytosolic and mitochondrial domains, incorporates key metabolic species and processes associated with energy transfer. Simulation of metabolic responses to mild, moderate and severe ischaemia in large animals showed that mitochondrial NADH/NAD+ was rapidly reset to higher values in proportion to the reduced O2 delivery and myocardial oxygen consumption . Cytosolic NADH/NAD+, however, showed a biphasic response, with a sharp initial increase that was due to activation of glycogen breakdown and glycolysis, and corresponded with lactate production. Whereas the rate of glycolysis and the malate–aspartate shuttle had a significant effect on the cytosolic NADH/NAD+, their effects on the mitochondrial NADH/NAD+ were minimal. In summary, model simulations of the metabolic response to ischaemia showed that mitochondrial NADH/NAD+ is primarily determined by O2 consumption, while cytosolic NADH/NAD+ is largely a function of glycolytic flux during the initial phase, and is determined by mitochondrial NADH/NAD+ and the malate–aspartate shuttle during the steady state.

HNO Enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization

AIMS Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. RESULTS Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. INNOVATION HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. CONCLUSIONS PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

Effects of Regional Mitochondrial Depolarization on Electrical Propagation Implications for Arrhythmogenesis

Background—Sudden cardiac death often involves arrhythmias triggered by metabolic stress. Loss of mitochondrial function is thought to contribute to the arrhythmogenic substrate, but how mitochondria contribute to uncoordinated electrical activity is poorly understood. It has been proposed that the formation of metabolic current sinks, caused by the nonuniform collapse of mitochondrial inner membrane potential (&Dgr;&PSgr;m), contributes to re-entrant arrhythmias because &Dgr;&PSgr;m depolarization is tightly coupled to the activation of sarcolemmal ATP-sensitive K+ channels, hastening action potential repolarization and shortening the refractory period. Methods and Results—Here, we use computational and experimental methods to investigate how &Dgr;&PSgr;m instability can induce re-entrant arrhythmias. We develop the first tissue-level model of cardiac electrical propagation incorporating cellular electrophysiology, excitation–contraction coupling, mitochondrial energetics, and reactive oxygen species balance. Simulations show that re-entry and fibrillation can be initiated by regional &Dgr;&PSgr;m loss because of the disparity of refractory periods inside and outside the metabolic sink. Computational results are compared with the effects of a metabolic sink generated experimentally by local perfusion of a mitochondrial uncoupler in a monolayer of cardiac myocytes. Conclusions—The results demonstrate that regional mitochondrial depolarization triggered by oxidative stress activates sarcolemmal ATP-sensitive K+ currents to form a metabolic sink. Consequent shortening of the action potential inside, but not outside, the sink increases the propensity for re-entry. &Dgr;&PSgr;m recovery during pacing can lead to novel mechanisms of ectopic activation. The findings highlight the importance of mitochondria as potential therapeutic targets for sudden death associated with cardiovascular disease.

Inhibiting Na+/K+ ATPase Can Impair Mitochondrial Energetics and Induce Abnormal Ca2+ Cycling and Automaticity in Guinea Pig Cardiomyocytes

Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na+/K+ ATPase (NKA), which raises [Na+]i and attenuates Ca2+ extrusion via the Na+/Ca2+ exchanger (NCX), causing [Ca2+]i elevation. The resulting [Ca2+]i accumulation further enhances Ca2+-induced Ca2+ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca2+ retention (thus suppressed reactive oxygen species (ROS) scavenging) but also enhanced oxidative phosphorylation (thus increased ROS production) during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na+/Ca2+ exchanger, but not enhancing of Ca2+ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca2+ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.

Stromal interaction molecule 1 is essential for normal cardiac homeostasis through modulation of ER and mitochondrial function

The endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) has been implicated as a key mediator of store-dependent and store-independent Ca(2+) entry pathways and maintenance of ER structure. STIM1 is present in embryonic, neonatal, and adult cardiomyocytes and has been strongly implicated in hypertrophic signaling; however, the physiological role of STIM1 in the adult heart remains unknown. We, therefore, developed a novel cardiomyocyte-restricted STIM1 knockout ((cr)STIM1-KO) mouse. In cardiomyocytes isolated from (cr)STIM1-KO mice, STIM1 expression was reduced by ∼92% with no change in the expression of related store-operated Ca(2+) entry proteins, STIM2, and Orai1. Immunoblot analyses revealed that (cr)STIM1-KO hearts exhibited increased ER stress from 12 wk, as indicated by increased levels of the transcription factor C/EBP homologous protein (CHOP), one of the terminal markers of ER stress. Transmission electron microscopy revealed ER dilatation, mitochondrial disorganization, and increased numbers of smaller mitochondria in (cr)STIM1-KO hearts, which was associated with increased mitochondrial fission. Using serial echocardiography and histological analyses, we observed a progressive decline in cardiac function in (cr)STIM1-KO mice, starting at 20 wk of age, which was associated with marked left ventricular dilatation by 36 wk. In addition, we observed the presence of an inflammatory infiltrate and evidence of cardiac fibrosis from 20 wk in (cr)STIM1-KO mice, which progressively worsened by 36 wk. These data demonstrate for the first time that STIM1 plays an essential role in normal cardiac function in the adult heart, which may be important for the regulation of ER and mitochondrial function.

Role of the Malate-Aspartate Shuttle on the Metabolic Response to Myocardial Ischemia

The malate-aspartate (M-A) shuttle provides an important mechanism to regulate glycolysis and lactate metabolism in the heart by transferring reducing equivalents from cytosol into mitochondria. However, experimental characterization of the M-A shuttle has been incomplete because of limitations in quantifying cytosolic and mitochondrial metabolites. In this study, we developed a multi-compartment model of cardiac metabolism with detailed presentation of the M-A shuttle to quantitatively predict non-observable fluxes and metabolite concentrations under normal and ischemic conditions in vivo. Model simulations predicted that the M-A shuttle is functionally localized to a subdomain that spans the mitochondrial and cytosolic spaces. With the onset of ischemia, the M-A shuttle flux rapidly decreased to a new steady state in proportion to the reduction in blood flow. Simulation results suggest that the reduced M-A shuttle flux during ischemia was not due to changes in shuttle-associated enzymes and transporters. However, there was a redistribution of shuttle-associated metabolites in both cytosol and mitochondria. Therefore, the dramatic acceleration in glycolysis and the switch to lactate production that occur immediately after the onset of ischemia is mediated by reduced M-A shuttle flux through metabolite redistribution of shuttle associated species across the mitochondrial membrane.