Impact of sarcoplasmic reticulum calcium release on calcium dynamics and action potential morphology in human atrial myocytes: a computational study

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca2+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.     

Modulation of action potential by [Ca2+]i in modeled rat atrial and guinea pig ventricular myocytes

We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.     

Mathematical modeling mechanisms of arrhythmias in transgenic mouse heart overexpressing TNF-α

Transgenic mice overexpressing tumor necrosis factor-α (TNF-α mice) possess many of the features of human heart failure, such as dilated cardiomyopathy, impaired Ca(2+) handling, arrhythmias, and decreased survival. Although TNF-α mice have been studied extensively with a number of experimental methods, the mechanisms of heart failure are not completely understood. We created a mathematical model that reproduced experimentally observed changes in the action potential (AP) and Ca(2+) handling of isolated TNF-α mice ventricular myocytes. To study the contribution of the differences in ion currents, AP, Ca(2+) handling, and intercellular coupling to the development of arrhythmias in TNF-α mice, we further created several multicellular model tissues with combinations of wild-type (WT)/reduced gap junction conductance, WT/prolonged AP, and WT/decreased Na(+) current (I(Na)) amplitude. All model tissues were examined for susceptibility to Ca(2+) alternans, AP propagation block, and reentry. Our modeling results demonstrated that, similar to experimental data in TNF-α mice, Ca(2+) alternans in TNF-α tissues developed at longer basic cycle lengths. The greater susceptibility to Ca(2+) alternans was attributed to the prolonged AP, resulting in larger inactivation of I(Na), and to the decreased SR Ca(2+) uptake and corresponding smaller SR Ca(2+) load. Simulations demonstrated that AP prolongation induces an increased susceptibility to AP propagation block. Programmed stimulation of the model tissues with a premature impulse showed that reduced gap junction conduction increased the vulnerable window for initiation reentry, supporting the idea that reduced intercellular coupling is the major factor for reentrant arrhythmias in TNF-α mice.     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Control of Ca Release by Action Potential Configuration in Normal and Failing Murine Cardiomyocytes

Cardiomyocytes from failing hearts exhibit spatially nonuniform or dyssynchronous sarcoplasmic reticulum (SR) Ca²⁺ release. We investigated the contribution of action potential (AP) prolongation in mice with congestive heart failure (CHF) after myocardial infarction. AP recordings from CHF and control myocytes were included in a computational model of the dyad, which predicted more dyssynchronous ryanodine receptor opening during stimulation with the CHF AP. This prediction was confirmed in cardiomyocyte experiments, when cells were alternately stimulated by control and CHF AP voltage-clamp waveforms. However, when a train of like APs was used as the voltage stimulus, the control and CHF AP produced a similar Ca²⁺ release pattern. In this steady-state condition, greater integrated Ca²⁺ entry during the CHF AP lead to increased SR Ca²⁺ content. A resulting increase in ryanodine receptor sensitivity synchronized SR Ca²⁺ release in the mathematical model, thus offsetting the desynchronizing effects of reduced driving force for Ca²⁺ entry. A modest nondyssynchronous prolongation of Ca²⁺ release was nevertheless observed during the steady-state CHF AP, which contributed to increased time-to-peak measurements for Ca²⁺ transients in failing cells. Thus, dyssynchronous Ca²⁺ release in failing mouse myocytes does not result from electrical remodeling, but rather other alterations such as T-tubule reorganization.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Effects of deletion of muscle LIM protein on myocyte function

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.     

Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

The effects of overexpression of the Na+/Ca2+ exchanger on calcium regulation in hypertrophied mouse cardiac myocytes

In cardiac hypertrophy and failure it has been shown that the amount of Na/Ca exchanger protein can increase. Several studies have investigated this modification in overt heart failure. However, the role of Na/Ca exchanger overexpression during the development of hypertrophy is unknown. To address this question we investigated Ca2+ regulation in an early stage of cardiac hypertrophy before signs of heart failure occurred and evaluated the role of Na/Ca exchanger overexpression. Cardiac hypertrophy was induced by a constant infusion of angiotensin II (Ang, 1 microg/min/kg) via an osmotic pump for 14 days. Thereafter, ventricular myocytes from either wild type (NON) or transgenic mice overexpressing the Na/Ca exchanger (TR) were isolated. Myocytes were loaded with indo-1 AM or fluo-4 AM to monitor cytoplasmic [Ca2+] with all experiments performed at 37 degrees C. In myocytes exposed to Ang there was an increase in cell capacitance of more than 20% indicating cellular hypertrophy. Ca2+ transients were prolonged in hypertrophied NON myocytes but not in TR myocytes. Action potentials had a less negative plateau in TR myocytes. Sarcoplasmic reticulum (SR) Ca2+ content, measured using rapid caffeine application, was greater in TR myocytes but unaffected by hypertrophy. Ca2+ spark frequency was significantly greater in TR. Na/Ca exchanger overexpression prevented the prolongation of the Ca2+ transient observed in hypertrophy and maintained a similar SR Ca2+ leak suggesting a compensatory role in Ca2+ regulation in hypertrophied cardiac myocytes from transgenic mice. We suggest this compensatory effect is mediated by increased SR Ca2+ content and faster Ca2+ removal via the Na/Ca exchanger.     

Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.     

Simulation and mechanistic investigation of the arrhythmogenic role of the late sodium current in human heart failure

Heart failure constitutes a major public health problem worldwide. The electrophysiological remodeling of failing hearts sets the stage for malignant arrhythmias, in which the role of the late Na(+) current (I(NaL)) is relevant and is currently under investigation. In this study we examined the role of I(NaL) in the electrophysiological phenotype of ventricular myocytes, and its proarrhythmic effects in the failing heart. A model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of I(NaL). A sensitivity analysis of the model was performed and simulations of the pathological electrical activity of the cell were conducted. The proposed model for the human I(NaL) and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. The sensitivity analysis of the modulation of electrophysiological parameters of myocytes from failing hearts due to ion channels remodeling, revealed a role for I(NaL) in the prolongation of action potential duration (APD), triangulation of the shape of the AP, and changes in Ca(2+) transient. A mechanistic investigation of intracellular Na(+) accumulation and APD shortening with increasing frequency of stimulation of failing myocytes revealed a role for the Na(+)/K(+) pump, the Na(+)/Ca(2+) exchanger and I(NaL). The results of the simulations also showed that in failing myocytes, the enhancement of I(NaL) increased the reverse rate-dependent APD prolongation and the probability of initiating early afterdepolarizations. The electrophysiological remodeling of failing hearts and especially the enhancement of the I(NaL) prolong APD and alter Ca(2+) transient facilitating the development of early afterdepolarizations. An enhanced I(NaL) appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of [Ca(2+)](i) homeostasis of failing myocytes.     

Computer model of action potential of mouse ventricular myocytes

We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.     

Measurements of Ca2+ entry and sarcoplasmic reticulum Ca2+ content during the cardiac cycle in guinea pig and rat ventricular myocytes.

This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Inhibition of Na+/Ca2+ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences

The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 microM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.     

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes

The Ca2+ sensitivity of a population of isolated adult rat heart myocytes has been related to the Na+ content of the cells prior to Ca2+ exposure, and the intracellular free Ca2+ as reported by quin2 fluorescence when the cells are challenged with millimolar external Ca2+. Myocytes exposed to Ca2+ during quin2 loading show a resting intracellular free Ca2+ of 150 +/- 30 nM and retain the rod cell morphology of heart cells in situ. The myocytes take up Na+ and lose K+ when incubated in the cold in the absence of Ca2+. Large numbers of these rod-shaped, Na+-loaded myocytes hypercontract into grossly distorted round cell forms when exposed to physiological levels of Ca2+. The number of cells that hypercontract is proportional to the Na+ content of the cells prior to Ca2+ addition and can be directly related to the intracellular free Ca2+ concentration attained following Ca2+ addition. Fifty percent of the cells in a myocyte population hypercontract when the internal free Ca2+ concentration reported by quin2 reaches 400 nM and virtually all of the cells hypercontract when this value reaches 1 microM. The entry of Ca2+ into Na+-loaded myocytes is biphasic with one phase inhibited by Ca2+ channel blockade. This suggests that Ca2+ enters Na+-loaded myocytes by the Ca2+ channel as well as by Na+/Ca2+ exchange.