In silico identification of potential calcium dynamics and sarcomere targets for recovering left ventricular function in rat heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a complex disease associated with multiple co-morbidities, where impaired cardiac mechanics are often the end effect. At the cellular level, cardiac mechanics can be pharmacologically manipulated by altering calcium signalling and the sarcomere. However, the link between cellular level modulations and whole organ pump function is incompletely understood. Our goal is to develop and use a multi-scale computational cardiac mechanics model of the obese ZSF1 HFpEF rat to identify important biomechanical mechanisms that underpin impaired cardiac function and to predict how whole-heart mechanical function can be recovered through altering cellular calcium dynamics and/or cellular contraction. The rat heart was modelled using a 3D biventricular biomechanics model. Biomechanics were described by 16 parameters, corresponding to intracellular calcium transient, sarcomere dynamics, cardiac tissue and hemodynamics properties. The model simulated left ventricular (LV) pressure-volume loops that were described by 14 scalar features. We trained a Gaussian process emulator to map the 16 input parameters to each of the 14 outputs. A global sensitivity analysis was performed, and identified calcium dynamics and thin and thick filament kinetics as key determinants of the organ scale pump function. We employed Bayesian history matching to build a model of the ZSF1 rat heart. Next, we recovered the LV function, described by ejection fraction, peak pressure, maximum rate of pressure rise and isovolumetric relaxation time constant. We found that by manipulating calcium, thin and thick filament properties we can recover 34%, 28% and 24% of the LV function in the ZSF1 rat heart, respectively, and 39% if we manipulate all of them together. We demonstrated how a combination of biophysically based models and their derived emulators can be used to identify potential pharmacological targets. We predicted that cardiac function can be best recovered in ZSF1 rats by desensitising the myofilament and reducing the affinity to intracellular calcium concentration and overall prolonging the sarcomere staying in the active force generating state.     

Formation of Spatially Discordant Alternans Due to Fluctuations and Diffusion of Calcium

Spatially discordant alternans (SDA) of action potential duration (APD) is a phenomenon where different regions of cardiac tissue exhibit an alternating sequence of APD that are out-of-phase. SDA is arrhythmogenic since it can induce spatial heterogeneity of refractoriness, which can cause wavebreak and reentry. However, the underlying mechanisms for the formation of SDA are not completely understood. In this paper, we present a novel mechanism for the formation of SDA in the case where the cellular instability leading to alternans is caused by intracellular calcium (Ca) cycling, and where Ca transient and APD alternans are electromechanically concordant. In particular, we show that SDA is formed when rapidly paced cardiac tissue develops alternans over many beats due to Ca accumulation in the sarcoplasmic reticulum (SR). The mechanism presented here relies on the observation that Ca cycling fluctuations dictate Ca alternans phase since the amplitude of Ca alternans is small during the early stages of pacing. Thus, different regions of a cardiac myocyte will typically develop Ca alternans which are opposite in phase at the early stages of pacing. These subcellular patterns then gradually coarsen due to interactions with membrane voltage to form steady state SDA of voltage and Ca on the tissue scale. This mechanism for SDA is distinct from well-known mechanisms that rely on conduction velocity restitution, and a Turing-like mechanism known to apply only in the case where APD and Ca alternans are electromechanically discordant. Furthermore, we argue that this mechanism is robust, and is likely to underlie a wide range of experimentally observed patterns of SDA.     

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.     

Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.

There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture.     

Regulation of Ca2+ and electrical alternans in cardiac myocytes: role of CAMKII and repolarizing currents

Alternans of cardiac repolarization is associated with arrhythmias and sudden death. At the cellular level, alternans involves beat-to-beat oscillation of the action potential (AP) and possibly Ca(2+) transient (CaT). Because of experimental difficulty in independently controlling the Ca(2+) and electrical subsystems, mathematical modeling provides additional insights into mechanisms and causality. Pacing protocols were conducted in a canine ventricular myocyte model with the following results: 1) CaT alternans results from refractoriness of the sarcoplasmic reticulum Ca(2+) release system; alternation of the L-type calcium current has a negligible effect; 2) CaT-AP coupling during late AP occurs through the sodium-calcium exchanger and underlies AP duration (APD) alternans; 3) increased Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity extends the range of CaT and APD alternans to slower frequencies and increases alternans magnitude; its decrease suppresses CaT and APD alternans, exerting an antiarrhythmic effect; and 4) increase of the rapid delayed rectifier current (I(Kr)) also suppresses APD alternans but without suppressing CaT alternans. Thus CaMKII inhibition eliminates APD alternans by eliminating its cause (CaT alternans) while I(Kr) enhancement does so by weakening CaT-APD coupling. The simulations identify combined CaMKII inhibition and I(Kr) enhancement as a possible antiarrhythmic intervention.     

Spatially discordant alternans in cardiomyocyte monolayers

Repolarization alternans is a harbinger of sudden cardiac death, particularly when it becomes spatially discordant. Alternans, a beat-to-beat alternation in the action potential duration (APD) and intracellular Ca (Cai), can arise from either tissue heterogeneities or dynamic factors. Distinguishing between these mechanisms in normal cardiac tissue is difficult because of inherent complex three-dimensional tissue heterogeneities. To evaluate repolarization alternans in a simpler two-dimensional cardiac substrate, we optically recorded voltage and/or Cai in monolayers of cultured neonatal rat ventricular myocytes during rapid pacing, before and after exposure to BAY K 8644 to enhance dynamic factors promoting alternans. Under control conditions (n = 37), rapid pacing caused detectable APD alternans in 81% of monolayers, and Cai transient alternans in all monolayers, becoming spatially discordant in 62%. After BAY K 8644 (n = 28), conduction velocity restitution became more prominent, and APD and Cai alternans developed and became spatially discordant in all monolayers, with an increased number of nodal lines separating out-of-phase alternating regions. Nodal lines moved closer to the pacing site with faster pacing rates and changed orientation when the pacing site was moved, as predicted for the dynamically generated, but not heterogeneity-based, alternans. Spatial APD gradients during spatially discordant alternans were sufficiently steep to induce conduction block and reentry. These findings indicate that spatially discordant alternans severe enough to initiate reentry can be readily induced by pacing in two-dimensional cardiac tissue and behaves according to predictions for a predominantly dynamically generated mechanism.     

Model of mechanical alternans in the mammalian myocardium

A model is proposed to elucidate the cause and mechanism of mechanical alternans in cardiac muscle in terms of discrete calcium movements. Mechanical alternans, the cause of which lies within the borders of excitation-contraction-coupling (ECC), is analyzed. In this case, the "input" of the ECC system (the action potentials and intervals) is constant while the "output" (contractile force) oscillates between two constant values, indicating that the system has a "memory" with two "internal states". It is proposed that these two "states" are associated with a part of the sarcoplasmic reticulum ("releasable terminal") containing the readily releasable calcium. A mechanism of "calcium-concentration-dependent threshold" is suggested to govern the "release function", i.e. the release of calcium from the "releasable terminal" to the myofilaments. The "release function" is analyzed in both the linear and the non-linear cases and its implication on the initiation of sustained and transient mechanical alternans are described. The dependence of mechanical alternans on a disturbance is also explained. The model response resembles the experimental observations of mechanical alternans in mammalian myocardium in the following manners: abrupt transition from low to high heart rates, slow progressive acceleration of rate, variations in persistence at subthreshold rates, effect of premature and delayed beat following the small and large beats, restitution curves, and transient mechanical alternans initiated by a delayed beat.     

Inferring the cellular origin of voltage and calcium alternans from the spatial scales of phase reversal during discordant alternans

Beat-to-beat alternation of the action potential duration (APD) in paced cardiac cells has been linked to the onset of lethal arrhythmias. Both experimental and theoretical studies have shown that alternans at the single cell level can be caused by unstable membrane voltage (V(m)) dynamics linked to steep APD-restitution, or unstable intracellular calcium (Ca) cycling linked to high sensitivity of Ca release from the sarcoplasmic reticulum on sarcoplasmic reticulum Ca load. Identifying which of these two mechanisms is the primary cause of cellular alternans, however, has remained difficult since Ca and V(m) are bidirectionally coupled. Here, we use numerical simulations of a physiologically detailed ionic model to show that the origin of alternans can be inferred by measuring the length scales over which APD and Ca(i) alternans reverse phase during spatially discordant alternans. The main conclusion is that these scales are comparable to a few millimeters and equal when alternans is driven by APD restitution, but differ markedly when alternans is driven predominantly by unstable Ca cycling. In the latter case, APD alternans still reverses phase on a millimeter tissue scale due to electrotonic coupling, while Ca alternans reverses phase on a submillimeter cellular scale. These results show that experimentally accessible measurements of Ca(i) and V(m) in cardiac tissue can be used to shed light on the cellular origin of alternans.     

Myocardial calcium signalling and arrhythmia pathogenesis

Myocardial calcium signalling is a vital component of the normal physiological function of the heart. Key amongst the many roles calcium plays is its use as the primary signalling component of excitation-contraction coupling, the intracellular process that links cardiomyocyte depolarisation to contraction. Defective cellular calcium handling, due to abnormalities of the various components which mediate and control excitation-contraction coupling, is widely recognised as a significant patho-physiological event in the contractile dysfunction of the failing heart. In addition, similar defects also appear to be increasingly recognised as mediators of certain forms of cardiac arrhythmias. Such defects include single gene defects in excitation-contraction coupling components that lead to inherited sudden death arrhythmia syndromes. Alternatively, arrhythmogenesis occurring within the context of acquired cardiac disease, in particular heart failure, also appears to be highly dependent on abnormal calcium homeostasis. In this article we review the defects in cardiomyocyte calcium homeostasis that lead to particular pro-arrhythmogenic phenomena and discuss recent insights gained into a variety of inherited and acquired arrhythmia syndromes that appear to involve defective calcium signalling as a central component of their patho-physiology. Potential opportunities for new anti arrhythmic therapeutic strategies based on these recent insights are also discussed.     

Melanocyte-keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes

Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte-keratinocyte co-cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a 'ligand-receptor' type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.     

Metabolic stages, mitochondria and calcium in hypoxic/ischemic brain damage

Cerebral hypoxia/ischemia leads to mitochondrial dysfunction due to lack of oxygen leaving the glycolytic metabolism as a main pathway for ATP production. Inhibition of mitochondrial respiration thus triggers generation of lactate and hydrogen ions (H+), and furthermore dramatically reduces ATP generation leading to disregulation of cellular ion metabolism with subsequent intracellular calcium accumulation. Upon reperfusion, when mitochondrial dysfunction is (at least partially) reversed by restoring cerebral oxygen supply, bioenergetic metabolism recovers and brain cells are able to re-institute their normal ionic homeostatic mechanisms. However, the initial restoration of normal mitochondrial function may be only transient and followed by a secondary, delayed perturbation of mitochondrial respiratory performance seen as a decrease in cellular ATP levels and known as "secondary energy failure". There have been several mechanisms considered responsible for delayed post-ischemic mitochondrial failure, the mitochondrial permeability transition (MPT) being one that is considered important. Although the amount of calcium available during early reperfusion in vivo is limited, relative to the amount needed to trigger the MPT in vitro; the additional intracellular conditions (of acidosis, high phosphate, and low adenine nucleotideae levels) prevailing during reperfusion, favor MPT pore opening in vivo. Furthermore, the cellular redistribution and/or changes in the intracellular levels of pro-apoptotic proteins can alter mitochondrial function and initiate apoptotic cell death. Thus, mitochondria seem play an important role in orchestrating cell death mechanisms following hypoxia/ischemia. However, it is still not clear which are the key mechanisms that cause mitochondrial dysfunction and lead ultimately to cell death, and which have more secondary nature to brain damage acting as aggravating factors.     

Simulation of intracellular $$\hbox {Ca}^{2+}$$ transients in osteoblasts induced by fluid shear stress and its application

Intracellular \(\hbox {Ca}^{2+}\) transient induced by fluid shear stress (FSS) plays an important role in mechanical regulation of osteoblasts, but the cellular mechanism remains incompletely understood. Here, we constructed a mathematical model combined with experiments to elucidate it. Our simulated and experimental results showed that it was the delay of membrane potential repolarization to produce the refractory period of FSS-induced intracellular calcium transients in osteoblasts. Moreover, the results also demonstrated that the amplitude of FSS-induced intracellular calcium transient is crucial to the proliferation, while its duration is critical to the differentiation, of osteoblasts. Overall, the present study provides a way to understand the cellular mechanism of intracellular calcium transients in osteoblast induced by FSS and explains some of related physiological events.     

Melanocyte–keratinocyte interaction induces calcium signalling and melanin transfer to keratinocytes

Physical contact between melanocytes and keratinocytes is a prerequisite for melanosome transfer to occur, but cellular signals induced during or after contact are not fully understood. Herein, it is shown that interactions between melanocyte and keratinocyte plasma membranes induced a transient intracellular calcium signal in keratinocytes that was required for pigment transfer. This intracellular calcium signal occurred due to release of calcium from intracellular stores. Pigment transfer observed in melanocyte–keratinocyte co‐cultures was inhibited when intracellular calcium in keratinocytes was chelated. We propose that a ‘ligand‐receptor’ type interaction exists between melanocytes and keratinocytes that triggers intracellular calcium signalling in keratinocytes and mediates melanin transfer.     

Ankyrin-B Protein in Heart Failure: IDENTIFICATION OF A NEW COMPONENT OF METAZOAN CARDIOPROTECTION

Ankyrins (ankyrin-R, -B, and -G) are adapter proteins linked with defects in metazoan physiology. Ankyrin-B (encoded by ANK2) loss-of-function mutations are directly associated with human cardiovascular phenotypes including sinus node disease, atrial fibrillation, ventricular tachycardia, and sudden cardiac death. Despite the link between ankyrin-B dysfunction and monogenic disease, there are no data linking ankyrin-B regulation with common forms of human heart failure. Here, we report that ankyrin-B levels are altered in both ischemic and non-ischemic human heart failure. Mechanistically, we demonstrate that cardiac ankyrin-B levels are tightly regulated downstream of reactive oxygen species, intracellular calcium, and the calcium-dependent protease calpain, all hallmarks of human myocardial injury and heart failure. Surprisingly, β(II)-spectrin, previously thought to mediate ankyrin-dependent modulation in the nervous system and heart, is not coordinately regulated with ankyrin-B or its downstream partners. Finally, our data implicate ankyrin-B expression as required for vertebrate myocardial protection as hearts deficient in ankyrin-B show increased cardiac damage and impaired function relative to wild-type mouse hearts following ischemia reperfusion. In summary, our findings provide the data of ankyrin-B regulation in human heart failure, provide insight into candidate pathways for ankyrin-B regulation in acquired human cardiovascular disease, and surprisingly, implicate ankyrin-B as a molecular component for cardioprotection following ischemia.     

Mechanisms of calcium transient and action potential alternans in cardiac cells and tissues

Alternation of cardiac action potential duration (APD) from beat to beat and concurrent alternation of the amplitude of the calcium transient are regarded as important arrhythmia mechanisms. These phenomena are causally interrelated and can be reliably evoked by an increase in beat frequency or by ischemia. The first part of this historical review deals with the physiology of APD alternans. Sections recounting the evolution of knowledge about calcium-activated ion currents and calcium transient alternans are interspersed among sections describing the growth of the so-called "restitution hypothesis," which involves time-dependent recovery of potassium channels (including their passage through pre-open states) as a function of diastolic interval. Major developments are generally in chronological order, but it is necessary to move back and forth between the two theories to respect the overall time line, which runs from about l965 to the present. The concluding two sections deal with the pathophysiology of calcium transient and APD alternans during ischemia, which may be the basis for out-of-hospital cardiac arrest during the initial stages of acute myocardial infarction.     

Cardiac cellular coupling and the spread of early instabilities in intracellular Ca2+

Recent experimental and modeling studies demonstrate the fine spatial scale, complex nature, and independent contribution of Ca²⁺ dynamics as a proarrhythmic factor in the heart. The mechanism of progression of cell-level Ca²⁺ instabilities, known as alternans, to tissue-level arrhythmias is not well understood. Because gap junction coupling dictates cardiac syncytial properties, we set out to elucidate its role in the spatiotemporal evolution of Ca²⁺ instabilities. We experimentally perturbed cellular coupling in cardiac syncytium in vitro. Coupling was quantified by fluorescence recovery after photobleaching, and related to function, including subtle fine-scale Ca²⁺ alternans, captured by optical mapping. Conduction velocity and threshold for alternans monotonically increased with coupling. Lower coupling enhanced Ca²⁺ alternans amplitude, but the spatial spread of early (<2 Hz) alternation was the greatest under intermediate (not low) coupling. This nonmonotonic relationship was closely matched by the percent of samples exhibiting large-scale alternans at higher pacing rates. Computer modeling corroborated these experimental findings for strong but not weak electromechanical (voltage-Ca²⁺) coupling, and offered mechanistic insight. In conclusion, using experimental and modeling approaches, we reveal a general mechanism for the spatial spread of subtle cellular Ca²⁺ alternans that relies on a combination of gap-junctional and voltage-Ca²⁺ coupling.     

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.     

Voltage and Calcium Dynamics Both Underlie Cellular Alternans in Cardiac Myocytes

Cardiac alternans, a putative trigger event for cardiac reentry, is a beat-to-beat alternation in membrane potential and calcium transient. Alternans was originally attributed to instabilities in transmembrane ion channel dynamics (i.e., the voltage mechanism). As of this writing, the predominant view is that instabilities in subcellular calcium handling are the main underlying mechanism. That being said, because the voltage and calcium systems are bidirectionally coupled, theoretical studies have suggested that both mechanisms can contribute. To date, to our knowledge, no experimental evidence of such a dual role within the same cell has been reported. Here, a combined electrophysiological and calcium imaging approach was developed and used to illuminate the contributions of voltage and calcium dynamics to alternans. An experimentally feasible protocol, quantification of subcellular calcium alternans and restitution slope during cycle-length ramping alternans control, was designed and validated. This approach allows simultaneous illumination of the contributions of voltage and calcium-driven instability to total cellular instability as a function of cycle-length. Application of this protocol in in vitro guinea-pig left-ventricular myocytes demonstrated that both voltage- and calcium-driven instabilities underlie alternans, and that the relative contributions of the two systems change as a function of pacing rate.     

Voltage and Calcium Dynamics Both Underlie Cellular Alternans in Cardiac Myocytes

Cardiac alternans, a putative trigger event for cardiac reentry, is a beat-to-beat alternation in membrane potential and calcium transient. Alternans was originally attributed to instabilities in transmembrane ion channel dynamics (i.e., the voltage mechanism). As of this writing, the predominant view is that instabilities in subcellular calcium handling are the main underlying mechanism. That being said, because the voltage and calcium systems are bidirectionally coupled, theoretical studies have suggested that both mechanisms can contribute. To date, to our knowledge, no experimental evidence of such a dual role within the same cell has been reported. Here, a combined electrophysiological and calcium imaging approach was developed and used to illuminate the contributions of voltage and calcium dynamics to alternans. An experimentally feasible protocol, quantification of subcellular calcium alternans and restitution slope during cycle-length ramping alternans control, was designed and validated. This approach allows simultaneous illumination of the contributions of voltage and calcium-driven instability to total cellular instability as a function of cycle-length. Application of this protocol in in vitro guinea-pig left-ventricular myocytes demonstrated that both voltage- and calcium-driven instabilities underlie alternans, and that the relative contributions of the two systems change as a function of pacing rate.