Cardiovascular Research

Cardiovascular Research 2007 73(1):101-110; doi:10.1016/j.cardiores.2006.10.028

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Copyright © 2006, European Society of Cardiology

Shock-induced changes of Ca_i²⁺ and V_m in myocyte cultures and computer model: Dependence on the timing of shock application

Vidya Raman, Andrew E. Pollard and Vladimir G. Fast^*

Department of Biomedical Engineering, University of Alabama at Birmingham, 1670 University Blvd, VH B126, Birmingham, Alabama 35294, United States

^* Corresponding author. Tel.: +1 205 975 2119; fax: +1 205 975 4720. Email address: fast{at}crml.uab.edu

Received 21 May 2006; revised 15 October 2006; accepted 31 October 2006

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
Objectives: Responses of Ca_i²⁺ to electrical shocks are believed to be important in defibrillation but measurements of shock-induced Ca_i²⁺ changes during different phases of the action potential (AP) are lacking. The effects of shocks on Ca_i²⁺ and V_m were investigated in geometrically defined cell cultures and in a computer model.

Methods: Uniform-field shocks (E=10.4±0.9 V/cm) were applied 15–300 ms after AP upstroke in strands of cultured neonatal rat myocytes. Optical mapping was used to measure shock-induced Ca_i²⁺ and V_m changes. A rat ionic model was used to elucidate ionic mechanisms of Ca_i²⁺ responses.

Results: In experiments and simulations, shocks applied with short delays (15–40 ms) caused a transient decrease of Ca_i²⁺ at sites of both V⁺_m and V^–_m. Simulations indicated that the Ca_i²⁺ decrease at V⁺_m sites was caused by reversed outward flow of L-type Ca²⁺ current (I_CaL), while the Ca_i²⁺ decrease at V^–_m sites was due to the NaCa exchanger (NCX). At intermediate delays (40–150 ms), shocks caused a Ca_i²⁺ decrease at sites of V^–_m and an increase at sites of V⁺_m. Simulations indicated that the Ca_i²⁺ increase at V⁺_m sites was caused by transient reactivation of I_CaL combined with a reverse-mode operation of NCX. Shocks applied at long delays (150–300 ms) caused a Ca_i²⁺ increase at V⁺_m and no change at V^–_m sites.

Conclusion: Effects of shocks on Ca_i²⁺ depend on the timing of shock application. Shocks applied during the early AP cause a transient Ca_i²⁺ decrease, while later in AP shocks induce a Ca_i²⁺ increase at sites of V⁺_m. Shock-induced Ca_i²⁺ changes in different AP phases are primarily determined by combination of I_CaL and NCX.

KEYWORDS Calcium; Computer modeling; Defibrillation; Mapping; Membrane potential

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
The outcome of defibrillation depends on shock-induced changes in membrane potential (V_m) and, possibly, changes in intracellular calcium concentration (Ca_i²⁺). Numerous studies were devoted to measurements of spatio-temporal shock-induced V_m changes (V_m) using optical mapping techniques. It is well established that shocks produce highly non-uniform and time-dependent V_m changes with areas of positive, negative or zero polarizations in different regions of the heart [1–5]. The contributions of Ca_i²⁺ changes to defibrillation outcomes have received little attention. Based on early studies, it was suggested that strong shocks may produce Ca_i²⁺ overload, which can cause post-shock arrhythmias and defibrillation failure [6]. It was also proposed that relatively weak shocks applied during fibrillation may raise Ca_i²⁺ level and thus prevent the loss of cardiac contractility observed after defibrillation [7,8]. In a recent study, the effects of shocks on Ca_i²⁺ and V_m were measured in cultured cell monolayers using optical mapping technique [9]. It was found that shocks applied during the early AP phase caused a transient decrease in Ca_i²⁺ in areas of both positive and negative V_m. Similar Ca_i²⁺ changes were also observed in a computer model of rat ventricular myocytes [9]. These findings might help to understand the effects of shocks on cardiac tissue but they are limited to the initial AP phase; data on Ca_i²⁺ changes during the later AP phases are presently lacking. Therefore, the goal of this study was to measure the effects of shocks on Ca_i²⁺ and V_m during different AP phases Experiments were performed in patterned-growth cultures of neonatal rat myocytes that allow Ca_i²⁺ and V_m mapping at high spatial resolution as well as the control of tissue geometry. In addition, computer simulations in ionic model of rat ventricular myocytes were carried out to provide insight into alterations of sarcolemmal currents and intracellular Ca_i²⁺ handling that may underlie the experimental observations.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
2.1. Cell cultures
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Linear cell strands (width0.8 mm) were grown using neonatal myocytes from 1 to 2 day old rats as described previously [9,10]. Cell cultures were incubated in UltraCulture medium (Fisher) supplemented with 20 µg/ml vitamin B₁₂, antibiotics, 0.1 mmol/L of bromodeoxyuridine and 1 µmol/L of epinephrine at 37 °C in a humidified atmosphere containing 4% CO₂. During experiments, cell cultures were superfused with Hank's balanced salt solution (pH=7.4, temperature=35–36 °C). Measurements were made between the fourth and sixth days in culture.

2.2. Optical mapping of V_m and Ca_i²⁺
Changes in V_m and Ca_i²⁺ were measured using optical mapping technique [9]. Cells were first stained with 5 µmol/L of low-affinity Ca_i²⁺-sensitive dye Fluo-4FF for 45 min and then with 4.5 µmol/L of V_m-sensitive dye RH-237 for 7 min. To reduce leakage of Fluo-4FF, 1 mmol/L of probenecid was added to staining and perfusion solutions [11].

Ca_i²⁺ and V_m changes were measured sequentially using different optical filters. For V_m measurements, excitation and emission filters were 560/55 nm and >650 nm, respectively. For Ca_i²⁺ measurements, the respective filters were 480/40 nm and 530/50 nm. Emitted fluorescence was measured using a 16x16-photodiode array (Hamamatsu) and a microscopic mapping system [12] at a sampling rate of 2–5 kHz/channel and spatial resolution of 55 or 110 µm/diode.

Cells were paced at 500-ms cycle length using a bipolar electrode. Rectangular uniform-field shocks (duration=10 ms) were delivered via two platinum plate electrodes. Field strength was measured using a bipolar electrode placed near the recording site. It was adjusted to 10 V/cm to produce the largest Ca_i²⁺ changes during the early AP phase [9]. The delay between shock application and AP upstroke was varied between 15 and 300 ms. To check for data reproducibility, control V_m and Ca_i²⁺ recordings were made before and after shock measurements. Both V_m and Ca_i²⁺ measurements were highly reproducible. To reduce dye bleaching and phototoxicity, the duration of recordings was limited to 500 ms and the number of recordings per site was restricted to ten.

Action potential amplitude (APA) and Ca_i²⁺ transient amplitude (ACa) were measured as differences between resting and peak levels of corresponding signals. Activation time was measured at the 50% level of APA. Only those cell strands that had average conduction velocity of >20 cm/s and showed no major discontinuities in activation spread and V_m distribution were selected for analysis in order to ensure uniformity of tissue responses. A shock-induced V_m was measured as the difference between linear fit of the pre-shock V_m and the V_m magnitude 1 ms before the shock end and normalized by the APA [13]. A shock-induced Ca_i²⁺ was measured as the difference between the shock and control recordings and normalized by the amplitude of the control ACa [9]. To reduce errors in determination of V_m and Ca_i²⁺ caused by motion artifact, V_m and Ca_i²⁺ signals were spatially averaged along the strand axis [11]. Such errors were further reduced by the fitting and subtraction procedure described above. Data were expressed as mean±SD.

2.3. Computer modeling
Computer simulations were performed in a one-dimensional monodomain cable model based on ionic model of adult rat endocardial ventricular myocytes [14]. The Ca²⁺-handling system in this model describes the ion flow between the extracellular space and three intracellular pools: cytoplasm, subspace and sarcoplasmic reticulum (SR). The flow of Ca²⁺ ions is carried by L-type Ca²⁺ current (I_CaL), NaCa exchange current, sarcolemmal pump current, SR release and uptake as well as intracellular binding by proteins such as troponin. The duration of Ca_i²⁺ transients in the rat ventricular model [14] (400 ms) is much larger than in neonatal rat myocyte cultures (CaD₈₀140 ms) [9]. To shorten the duration of Ca_i²⁺ transients in the model, parameters of SR and troponin binding were slightly modified as described previously [9].

In order to analyze contributions of various Ca²⁺ handling pathways in shock-induced Ca_i²⁺ changes, we calculated relative rates of Ca²⁺ concentration changes associated with these pathways. In accordance with the original model formulation, [14] these variables are called "fluxes". Formulations for relevant fluxes are given in the Supplement. The cytoplasmic Ca²⁺ concentration (Ca_i²⁺) is determined by several fluxes among which the most important ones are the transfer flux (J_xfer) carrying ions between the cytoplasm and the subspace, the flux due to NaCa exchanger (J_NaCa), the flux due to SR uptake (J_up) and the flux due to Ca²⁺ binding by troponin (J_trpn). The subspace Ca²⁺ concentration (Ca_ss²⁺) is determined by the flux due to I_CaL (J_CaL), the flux due to SR Ca²⁺ release (J_rel) and the transfer flux (J_xfer). The advantage of using fluxes is that they allow direct comparison of qualitatively different Ca²⁺-handling pathways such as L-type Ca²⁺ current and troponin binding. This approach therefore represents a refinement of the earlier work [9] in which analysis focused on I_CaL changes.

The cable length was 0.96 mm. Model parameters were uniform along the cable. The following parameters were used: specific membrane capacitance 1 µF/cm², intracellular resistivity 500 cm, surface-to-volume ratio 6250 cm^–1. The cable was divided into 32 nodes with a length of 30 µm and the cable equation was solved numerically using previously published procedures [15] with temporal integration step of 0.1 µs. Each node was stimulated simultaneously. After a delay of 15–300 ms, a current was injected at one end of the cable and the same current was withdrawn at the other end. Such current injection in a monodomain model is equivalent to applying a uniform-field shock in a model with extracellular space. The pulse duration was 10 ms. The current strength was chosen to generate V_m with magnitudes similar to those observed experimentally during the early AP phase.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
3.1. Shock-induced Ca_i²⁺and V_m in cell cultures
Experiments were carried out in a total of 61 strands from 16 cell monolayers and 10 cultures. The average conduction velocity was 28±4 cm/s, which is similar to previous publications [12,16,17]. Uniform-field shocks with strength of 10.4±0.9 V/cm were applied 15.5±0.9 to 299±6 ms after the AP upstroke. The magnitude and the distribution pattern of shock-induced V_m and Ca_i²⁺ were strongly dependent on the timing of shock application. Based on the combination of Ca_i²⁺ and V_m changes, shock delays were classified into three categories: short delays (15–45 ms), intermediate delays (45–150 ms) and long delays (150–300 ms).

Fig. 1 shows results of typical measurements when shocks were applied with a short delay. Similar to the previous study [9], a shock applied 15 ms after the AP upstroke produced negatively asymmetric V_m (V^–_m/V⁺_m=3.2) and transient Ca_i²⁺ decrease in areas of both V^–_m and V⁺_m (Panels A, B). The magnitude of Ca_i²⁺ decrease was largest at the anodal edge of the strand where Ca_i²⁺ value averaged along the strand edge was –19% ACa. At the cathodal edge, the average Ca_i²⁺ change was –10% ACa. Between the edges, Ca_i²⁺ magnitude decreased reaching approximately 4% near the strand center (Panel C).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Vm and Cai2+ induced by shocks applied during the early AP phase (delay from the AP upstroke 15 ms). Shock strength 10 V/cm. Panel A, isopotential maps of Vm and Cai2+ distributions at the shock end. Panel B, optical recordings of Vm and Cai2+ in control and during shock application. The numbers correspond to the recording sites shown in Panel A. Panel C, spatial profiles of shock-induced Vm and Cai2+ across the cell strand. Locations of measurements are shown by grids in Panel A.

 
Fig. 2A, B shows V_m and Ca_i²⁺ changes when a shock was applied with an intermediate delay (90 ms). At his delay, the shock induced a larger V⁺_m and a smaller V^–_m than at the shorter delay. This resulted in reduction of V^–_m/V⁺_m from 3.2 to 1.5. Similar to the shorter delay, the shock caused Ca_i²⁺ decrease at the anodal strand edge (Panel B). At the cathodal edge, however, the shock caused a small Ca_i²⁺ increase.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Shock-induced Vm and Cai2+ during shocks applied at intermediate and long delays. Panels A and B, Vm and Cai2+ recordings (A) and profiles of Vm and Cai2+ (B) during shock application with 90-ms delay. Panels C and D, Vm/Cai2+ traces (A) and spatial Vm/Cai2+ profiles during shock application with 150-ms delay. Recording locations are shown in Fig. 1.

 
Fig. 2C, D shows V_m and Ca_i²⁺ changes produced by shocks applied with a long delay (150 ms). Such shock produced nearly symmetric V_m (V^–_m/V⁺_m=1.2). Ca_i²⁺ was changed little during the shock in the V^–_m area and slightly increased in the V⁺_m area. Across the strand, Ca_i²⁺ increase was non-uniform reaching its maximal value of 8% near the strand middle (Panel D). Right after the shock, a new action potential and a new Ca_i²⁺ transient with reduced amplitude and duration were generated. At the 300-ms delay (not shown), the asymmetry of shock-induced V_m was reversed from negative to positive with V⁺_m becoming slightly larger than V^–_m (V^–_m/V⁺_m=0.87). The shock had only a small effect on Ca_i²⁺ at the strand edges. Similar to the shorter delay, Ca_i²⁺ was largest (50%) near the strand middle where V_m amplitude was 60% APA. After the shock, a new Ca_i²⁺ transient with normal amplitude was generated.

Qualitatively similar results were obtained in other experiments. Each shock delay was examined in 8–15 strands. Shocks applied with delays of 15.5±0.9 and 30.8±1.3 ms decreased Ca_i²⁺ at both anodal and cathodal strand edges (Fig. 3C); shock-induced polarizations were strongly asymmetric V_m with larger negative than positive V_m (Fig. 3A, B). For delays of 45.3±0.9, 60±1, 75±1, 90±1.4 and 120±1 ms, shocks decreased Ca_i²⁺ at the anodal edge but increased it at the cathodal edge. At longest delays of 149±7 and 299±6 ms, shocks caused small Ca_i²⁺ increases at the cathodal edge but no Ca_i²⁺ changes at the anodal edge. With increasing the shock delay, V⁺_m gradually increased and V^–_m decreased which resulted in a reversal of V_m asymmetry from negative to positive at a shock delay of approximately 150 ms (Fig. 3B). At the same time, the sum of V⁺_m and V^–_m remained nearly constant.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Dependences of shock-induced Vm and Cai2+ on the shock timing. Panel A, maximal positive and negative Vm. Panel B, sum and ratio of maximal negative and positive Vm. The dashed line corresponds to symmetric Vm. Panel C, Cai2+ at cathodal and anodal strand edges.

 
3.2. Shock-induced Ca_i²⁺ and V_m in the computer model
Fig. 4 illustrates effects of a shock applied after the early AP phase (delay 10 ms) in a 1-dimensional computer model. Qualitatively similar to cell cultures, the shock caused negatively asymmetric polarizations and Ca_i²⁺ decreases at sites of both V^–_m and V⁺_m (Panel A). These traces are similar to the ones published previously [9] and are presented here for comparison with the effects of shocks applied at longer delays. To appreciate how different Ca²⁺ handling pathways contributed to the observed Ca_i²⁺ changes, ion fluxes at the cathodal and anodal ends of the model were compared to fluxes obtained under control (no shock) conditions. Panel B shows the four main fluxes (J_xfer, J_up, J_NaCa, J_trpn) used to update Ca_i²⁺ in the ionic model [14]. Panel C shows J_CaL and J_rel combined with a scaled version of J_xfer (see Supplement), which were used to update Ca_ss²⁺. For simplicity, minor fluxes (J_BCa and J_CaP) and the SR pool of calcium ions are not shown. Panel D shows diagrams including the directions for these main fluxes as Ca²⁺ moved between the extracellular, SR, subspace and cytoplasmic compartments under control and during shock application.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Modeling of shock-induced Vm and Cai2+ for shocks applied in the early AP phase (delay 10 ms). Panel A, recordings of Vm and Cai2+ at the anodal and cathodal ends of the cable (sites 1 and 2, respectively) in control and during shock application. E, shock waveform. Dashed lines depict zero signal level. Panel B, cytoplasmic fluxes of Ca2+ ions. Jxfer, the flux between the cytoplasm and the subspace; JNaCa, the flux carried by the NaCa exchanger; Jup, the flux due to uptake by the SR; Jtrpn, the flux due to troponin binding. Panel C, subspace ion fluxes. JCaL, the flux due to L-type calcium current; Jrel, the flux due to SR release. Panel D, schematic diagram of main Ca2+ fluxes in control (left panel) and during the shock at the cathodal and anodal ends of the cable (middle and right panels, respectively). Double-line arrows depict dominant Ca2+ fluxes. Dashed red and blue circles depict dominant fluxes responsible for shock-induced Cai2+ changes at the sites of V+m and V–m, respectively.

 
In control conditions, membrane depolarization during the AP upstroke activated J_CaL (Panel C, top black trace) and elevated Ca_ss²⁺, which induced Ca²⁺ release from SR via J_rel (Panel C, bottom black trace). In these membrane equations, J_CaL is 2-times larger than J_rel. As J_CaL and J_rel combined to increase Ca_ss²⁺, that increase established a concentration gradient directed toward the cytoplasm that resulted in Ca²⁺ transfer into the cytoplasm via J_xfer (Panel B, top black trace). Membrane depolarization also caused Ca²⁺ entry into the cytoplasm via reverse-mode J_NaCa (Panel B, middle black trace), although the magnitude for J_NaCa was only 1/6 the magnitude of J_Jxfer. Ca²⁺ ions entering the cytoplasm that were not pumped into SR (J_up) or bound to troponin (J_trpn) caused Ca_i²⁺ to rise, forming the transient. In Panel D, double arrows are drawn for J_CaL, J_xfer and J_trpn to emphasize their dominant contributions to the Ca_i²⁺ transient during the control AP.

The shock-induced Ca_i²⁺ decrease observed at the V⁺_m site resulted primarily from outward J_CaL (Panel C, top red trace) when V_m was elevated above the I_CaL reversal potential. The J_rel at this location was briefly increased and then decreased (Panel C, bottom red trace). Both the outward J_CaL and the decreased J_rel contributed to the reduction of Ca_ss²⁺ but J_CaL was more important factor because the shock-induced change in J_CaL was 5 times larger than the change in J_rel. Removal of Ca²⁺ from the subspace reversed the normal subspace-cytoplasm Ca²⁺ gradient leading to Ca²⁺ outflow from the cytoplasm by J_xfer (Panel B, top red trace). The highly elevated V_m enhanced reverse-mode NCX such that J_NaCa (Panel B, middle red trace) brought more Ca²⁺ into the cytoplasm than in control. However, J_NaCa entry was not sufficient to counteract J_xfer outflow. SR uptake and troponin binding (Panel B, middle and bottom red traces) continued during the shock but these fluxes became smaller in comparison to control indicating that they were not the cause for the shock-induced Ca_i²⁺ decrease.

The Ca_i²⁺ decrease at the V^–_m site resulted primarily from shock-induced switch of NCX from reverse to forward mode and outflow of Ca²⁺ ions via J_NaCa (Panel B, middle blue trace). J_trpn and J_up maintained their direction but were significantly reduced in comparison to control, which indicates that they were not responsible for the shock-induced Ca_i²⁺ decrease. Negative polarization also rapidly inactivated J_CaL (Panel C, top blue trace) and slowed J_xfer (Panel B, top blue trace), contributing to Ca_i²⁺ decrease.

Fig. 5 shows the effects of shocks applied at an intermediate delay of 75 ms. Similar to experiments, the shock caused an increase of Ca_i²⁺ at V⁺_m side and a small Ca_i²⁺ decrease at V^–_m side (Panel A). In contrast to cell cultures, however, V_m asymmetry reversed with V⁺_m becoming larger than V^–_m. The time course of Ca_i²⁺ change at the V⁺_m site was biphasic displaying first Ca_i²⁺ elevation (phase I) and then decline (phase II). These biphasic Ca_i²⁺ changes were paralleled with corresponding changes in J_CaL (Panel C) and J_xfer (Panel B). In the first phase, J_CaL was reactivated due to V_m depolarization causing inflow of Ca²⁺ ions into the subspace and, via J_xfer, into the cytoplasm. At the same time, the shock also switched the NCX from forward to reverse mode causing inflow of Ca²⁺ via J_NaCa but this flux was much smaller than J_xfer. In the second phase, due to continuing V_m elevation past the I_CaL reversal potential, J_CaL switched its direction from inward to outward reversing the direction of J_xfer as well and causing Ca_i²⁺ to decrease.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Modeling of shock-induced Vm and Cai2+ for intermediate shock delay (75 ms). Panel A, recordings of Vm and Cai2+. Panels B and C, cytoplasmic and subspace Ca2+ fluxes, respectively. Panel D, schematic diagram of Ca2+ fluxes. Designations are the same as in Fig. 4.

 
At the V^–_m site, the shock did not affect J_CaL, J_rel and J_xfer, all of which remained inactivated (Panels B and C). The only significant difference from control was an increased Ca²⁺ outflow via forward J_NaCa (Panel B), which accounted for the Ca_i²⁺ decrease at this location. This Ca²⁺ decrease was partially offset by the reversed J_trpn due to ion dissociation from troponin.

A shock applied at a delay of 135 ms produced changes in V_m and Ca_i²⁺ (Fig. 6A) as well as ion fluxes (data not shown) that were qualitatively similar to those observed at the intermediate 75-ms delay. The positive V_m asymmetry was augmented with V⁺_m becoming approximately 4-fold larger than V^–_m. The shock caused a small Ca_i²⁺ increase at the V⁺_m side and small but perceptible Ca_i²⁺ decrease at the V^–_m side. These results are different from measurements in cell cultures where V_m were nearly symmetric and Ca_i²⁺ was negligible at the V^–_m side at comparable shock delays (Fig. 3). At the longest delay of 300 ms, a shock produced V_m and Ca_i²⁺ responses (Fig. 6B) that were qualitatively different from those ones observed at shorter delays. The main difference was that the anodal V_m (trace 1) exhibited a large positive deflection during the shock. This deflection was likely a new action potential caused by activation spread from the neighboring positively polarized region. Generation of this new AP caused elevation of Ca_i²⁺ in the V^–_m area towards the end of the shock. This effect was dependent on the shock strength. A two-fold increase of the shock strength resulted in elimination of the new AP (data not shown) and shock-induced V_m and Ca_i²⁺ became qualitatively similar to those one observed at the shorter 135-ms delay.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effects of shocks in Vm and Cai2+ in the model at long shock delays. Panel A, shock delay=135 ms. Panel B, shock delay=300 ms.

 
Fig. 7 presents a summary of shock effects on V_m and Ca_i²⁺ in the computer model. Shocks applied with delays of less than 40 ms caused negatively asymmetric V_m (Panel B) and Ca_i²⁺ decreases at sites of both V^–_m and V⁺_m (Panel C). With increasing shock delay, the V_m asymmetry became reversed (Panel B) due a parallel increase of V⁺_m and a decrease of V^–_m (Panel A). At the same time, the sum of V⁺_m and V^–_m remained nearly constant (Panel B). At long delays, shocks caused an increase of Ca_i²⁺ at the V⁺_m side whereas at the V^–_m side Ca_i²⁺ remained negative. The data for the longest 300-ms delay are not shown because at this shock delay a new action potential was generated in the area of negative V_m before the shock end.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Dependences of shock-induced Vm and Cai2+ on the shock delay in the model. Panel A, maximal positive and negative Vm measured at the cathodal and anodal cable ends. The horizontal dashed line corresponds to symmetric Vm. Panel B, sum and ratio of maximal negative and positive Vm. Panel C, Cai2+ at the cathodal and anodal cable ends. The vertical dashed line corresponds to the transition of cathodal Cai2+ from negative to positive.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
In this study, effects of shocks on Ca_i²⁺ and V_m were measured during different AP phases in myocyte cultures; computer simulations were used to provide insight into possible ionic mechanisms for the shock-induced Ca_i²⁺ changes. The main findings are as follows. (1) Shocks applied at short delays produced negatively asymmetric V_m; Ca_i²⁺ was decreased in areas of both V⁺_m and V^–_m. Simulations indicated that Ca_i²⁺ decrease in the V⁺_m area was primarily due to reversed I_CaL; Ca_i²⁺ decrease in the V^–_m area was mainly due to removal of ions by NCX. (2) At intermediate delays, shocks caused a small Ca_i²⁺ increase in V⁺_m area and a decrease in V^–_m area. Simulations indicated that V⁺_m-related Ca_i²⁺ increase was caused by transient reactivation of I_CaL combined with Ca²⁺ influx via reverse-mode NCX; the V^–_m-related Ca_i²⁺ decrease was due to the forward-mode NCX. (3) Shocks applied at long delays in cell cultures produced nearly symmetric V_m; they also caused a small Ca_i²⁺ increase in the V⁺_m area and no change in the V^–_m area. This was different from the computer model where V⁺_m became significantly larger than V^–_m and Ca_i²⁺ remained negative in the V^–_m area.

4.1. Phase dependence of shock-induced V_m
Understanding the mechanism of defibrillation requires the knowledge of shock effects on V_m during various AP phases. Several previous studies reported that shocks applied during the early AP produce negatively asymmetric V_m with V^–_m>V⁺_m [1,12,18–20]. It was also reported that shocks applied during the late repolarization phase in myocyte cultures elicited nearly symmetric V_m.[18] Results from cell cultures obtained in the present study are in agreement with these publications. Detailed measurements of shock-induced V_m during the AP indicate that with increasing shock delay the V_m asymmetry changed gradually; V_m became nearly symmetric at a shock delay of 150 ms and the V_m asymmetry became slightly positive at a delay of 300 ms (Fig. 3B). Qualitatively similar results were obtained in the computer model where negatively asymmetric V_m were observed at short delays, the V_m asymmetry was reduced and then reversed from negative to positive at longer delays. There were, however, quantitative differences between the experiments and the model. Thus, the degree of asymmetry at the shortest delays was substantially higher in experiments (V^–_m/V⁺_m3.0) than in the model (1.7). In addition, the V_m asymmetry in the model reversed at much shorter shock delays (40 ms, Fig. 7B) than in cell cultures and a much larger positive V_m asymmetry was achieved in the model at long delays (V^–_m/V⁺_m0.25 at 135-ms delay) than in cell cultures (0.9 at 300-ms delay). These differences indicate that the kinetics of ionic channels in the model is substantially different from that one in cell cultures.

4.2. Mechanisms of shock-induced Ca_i²⁺
Effects of shocks on Ca_i²⁺ are considered important for understanding post-shock arrhythmias and changes in cardiac contractility [6–8]. Previously, shock-induced Ca_i²⁺ changes were measured in cell cultures and analyzed in an ionic model during the early AP phase [9]. The present study extends this analysis to the whole action potential. It indicates that Ca_i²⁺ changes strongly depend on the timing of shock application. In agreement with the previous work [9], shocks applied during the early AP phase caused a transient decrease of Ca_i²⁺ in areas of both V⁺_m and V^–_m. Comparison of various ionic fluxes in the computer model performed here indicates that Ca_i²⁺ decrease at V⁺_m sites was primarily due to the outward flow of L-type Ca²⁺ current. The time window during which shocks caused Ca_i²⁺ to decrease was approximately 30 ms both in cell cultures and in the model (Figs. 3C and 7C). This is similar to the duration of the open state of I_CaL channels (Fig. 4C) supporting the role I_CaL in Ca_i²⁺ decrease at V⁺_m sites. Results of simulations also indicate that at V^–_m sites, Ca_i²⁺ decrease was caused by the removal of Ca²⁺ ions by NCX when I_CaL became inactivated.

Although experimental and modeling data on shock-induced Ca_i²⁺ measured during the early AP phase are in accordance with the previous publication [9], the interpretation of these data regarding the underlying ionic mechanisms for Ca_i²⁺ changes is somewhat different. This difference in interpretation concerns the role of troponin binding in Ca_i²⁺ decrease during shocks in the V^–_m area. Previously, the role of troponin binding was investigated by measuring the effect of disabling the binding during shock. Such blockade caused an increase in the Ca_i²⁺ level in comparison to the situation when the troponin binding was normal during shock application. These data were interpreted as an indication that troponin binding contributed to shock-induced Ca_i²⁺ decrease. This result, however, can be also explained by that troponin binding merely provided a passive sink for Ca²⁺ ions and elimination of this sink during a shock caused Ca_i²⁺ increase due to influx via other Ca²⁺-handling pathways. The analysis of the troponin-related Ca²⁺ flux (J_trpn) performed here is consistent with the second interpretation. It indicates that troponin binding was already significantly reduced during shocks in comparison to control conditions (Fig. 4B). Therefore, troponin binding was not the reason for the shock-induced Ca_i²⁺ decrease relative to control.

In contrast to the early phase of AP, it was found here that shocks applied at delays longer than 40 ms caused a transient Ca_i²⁺ increase at sites of V⁺_m in both cell cultures (Fig. 3C) and in the computer model (Fig. 7C). Examination of Ca²⁺ fluxes in the model indicated that such Ca_i²⁺ increase was primarily due to transient reactivation of I_CaL and, to a lesser extent, due to the reverse mode operation of NCX. The rapid switching of I_CaL from inactive state to inward flow and then to outward flow is explained by a rise of V_m from a negative level to the I_CaL activation threshold and then to the I_CaL reversal potential.

At V^–_m sites, shocks applied in cell cultures caused a transient Ca_i²⁺ decrease for delays longer than 40 ms and no change in Ca_i²⁺ at delays of 150 ms (Fig. 3C). Analysis of the shock effects on Ca²⁺ fluxes in the computer model indicated that Ca_i²⁺ decrease was caused mainly by ion efflux via forward NCX (Fig. 5B). At the shock onset, I_CaL, J_rel and J_xfer fluxes were inactive and remained unaffected during shocks. In contrast to cell cultures, anodal Ca_i²⁺ in the model remained slightly negative for all examined delays (Fig. 7C). This difference in Ca_i²⁺ response may be attributed to differences in the duration of Ca_i²⁺ transients between the model (CaD₈₀260 ms) and cell cultures (CaD₈₀140 ms). In the model, Ca_i²⁺ was still elevated at the moment of shock application with 150-ms delays. Because NCX is highly sensitive to Ca²⁺ concentration, shock-induced negative polarization maintained the efflux of Ca²⁺ ions by NCX. In cell cultures, however, NCX is likely to be inactive due to low Ca²⁺ concentration at the moment of shock application.

Shock-induced Ca_i²⁺ changes were implicated in the inotropic effect of low-energy shocks applied during defibrillation and alleviation of after-shock "pulse-less electrical activity" [7,8,21]. The results of the present study do not support this hypothesis. With the exception of shocks falling in diastole, the prevailing effect of shocks on cells was Ca_i²⁺ decrease in areas of both negative and positive V_m. Shocks caused Ca_i²⁺ elevation in the V⁺_m area at the end of AP but this effect was small and unlikely to cause a significant enhancement of cell contractility.

4.3. Limitations
Extrapolation of the results obtained in cultures of neonatal rat myocytes to other species and to the adult myocardium may be limited due to the species- and age-dependent differences in ionic currents and Ca_i²⁺ fluxes [22–27] that may result in differences of Ca_i²⁺ responses to electrical shocks. One such difference is the lower level of development of SR in neonatal cells [22]. In adult cells, SR plays a bigger role in Ca_i²⁺ regulation and, therefore, it may be more important in Ca_i²⁺ changes during shocks than in the neonatal cells.

Application of the ionic mechanisms of shock-induced Ca_i²⁺ changes inferred from the mathematical model may be also limited by the differences between the properties of the adult rat myocyte model [14] and neonatal cardiac cells. One such difference is the lack of T-tubules and, therefore, the subspace in neonatal myocytes. The main consequence of this difference is that calcium ions entering cells via I_CaL and SR release channels flow directly into the cytoplasm instead of passing through the subspace first. Although this difference may affect the quantitative aspects of Ca_i²⁺ kinetics, it is unlikely to change it qualitatively. Another difference is a significantly longer duration of Ca_i²⁺ transients in the model compared to neonatal rat myocytes, which was probably the cause for the differences in Ca_i²⁺ responses to shocks applied during the late AP phase. Although Ca_i²⁺ responses in cell cultures and in the model at long shock delays were quantitatively different, the analysis of Ca_i²⁺ changes in the model provides a valuable insight into ionic mechanisms of shock effects on Ca_i²⁺. Such understanding should become more complete when a more precise ionic model of neonatal rat ventricular cells becomes available.

	Appendix A. Supplementary data

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.10.028.

	Acknowledgements

 
This work was supported by NIH grants HL67748, HL67961 and AHA grant 0255025B. We thank Reuben Collins for help in the preparation of cell cultures.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Appendix A. Supplementary data References

 

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