Copyright © 2007, European Society of Cardiology
K201 modulates excitation–contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes
aInstitute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, UK
bFaculty of Biomedical & Life Sciences, University of Glasgow, UK
cDepartment of Cardiology and Pneumology, Georg-August-University Goettingen, D-37075, Goettingen, Germany
dDepartment of Cardiology and Pneumology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi, Japan
*Corresponding author. Faculty of Biomedical & Life Sciences, West Medical Building, Level 4, Glasgow, G12 8QQ, UK. Tel.: +44 141 330 6309; fax: +44 141 330 4612. g.smith{at}bio.gla.ac.uk
Received 19 March 2007; revised 15 June 2007; accepted 18 June 2007
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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Objectives The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown.
Methods The effect of K201 on excitation–contraction (E–C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca2+ measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes.
Results Acute application of <1 µmol/L K201 had no significant effect on E–C coupling. K201 at 1 µmol/L decreased Ca2+ transient amplitude (to 83±7%) without affecting ICa,L or the SR Ca2+ content. At 3 µmol/L K201 caused a larger reduction of Ca2+ transient amplitude (to 60±7%) with accompanying reductions in ICa,L amplitude (to 66±8%) and SR Ca2+ content (74±9%). Spontaneous SR Ca2+ release during diastole was induced by increasing intracellular [Ca2+]. At 1 µmol/L K201 reduced the frequency of spontaneous Ca2+ release. The effect of K201 on SR-mediated Ca2+ waves and Ca2+ sparks was examined in β-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 µmol/L) reduced the frequency and velocity of SR Ca2+ waves despite no change in SR Ca2+ content. At 3 µmol/L K201 completely abolished Ca2+ waves and reduced the SR Ca2+ content (to 
73%). K201 at 1 µmol/L reduced Ca2+ spark amplitude and frequency. Assays specific to SR Ca2+-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca2+ uptake and release.
Conclusions K201 modifies E–C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca2+ release during Ca2+ overload without significantly affecting Ca2+ transient amplitude.
KEYWORDS Calcium cycling/excitation–contraction coupling; Electrophysiology; Arrhythmias
This article is referred to in the Editorial by A.F. James (pages 199–201) in this issue.
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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The drug K201 (JTV-519) is a 1,4 benzothiazepine derivative which was found to be more effective than other Ca2+ channel antagonists at reducing Ca2+-induced myocardial damage [1]. While the drug is known to have multiple sites of action in the heart, recent work suggests that the beneficial effect of K201 during heart failure (HF) is mediated by actions on the ryanodine receptor (RyR2) complex [2,3]. In HF, abnormal function of RyR2 is thought to result in excessive diastolic Ca2+ release contributing to (a) impaired contractile function and (b) increased incidence of arrhythmias [3]. One theory for abnormal RyR2 function centres on the loss of an RyR2 accessory protein (FK-506 binding protein — FKBP12.6) [4]. A second related possibility is reduced RyR2 inter-domain interaction [5]. Previous studies suggest that K201: (a) restores the binding of FKBP12.6 to RyR2 [3] and/or (b) restores RyR2 inter-domain interaction [5]. To date, the effect of K201 on E–C coupling has only been examined in HF models or after disruption of RyR2-FKBP12.6 interaction.
This study is the first to investigate the effect of K201 on E–C coupling in cardiomyocytes isolated from normal hearts. Using both electrophysiology and confocal imaging, we demonstrate that K201 (1 µmol/L) reduces Ca2+ transient amplitude in normal rabbit cardiomocytes without changing L-type Ca2+ transient amplitude or SR Ca2+ content. When cardiomyocytes in Ca2+ overload were exposed to K201, the incidence of spontaneous diastolic SR Ca2+ release was reduced but the amplitude of the Ca2+ transient was unchanged. These actions of K201 are explained in terms of inhibitory effects on RyR2 and SR Ca2+-ATPase (SERCA).
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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2.1 Ventricular cardiomyocyte isolation
New Zealand White rabbits (2–2.5 kg) were euthanised by administration of an intravenous injection of 500 IU heparin together with an overdose of sodium pentobarbitone (100 mg kg–1). Hearts were removed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986, and conformed to the Guide for the Care and use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Rabbit ventricular cardiomyocytes were then isolated as previously described [6].
2.2 Electrophysiological measurements in rabbit cardiomyocytes
The isolated cardiomyocytes were superfused with a Krebs Hensliet solution (mmol/L): 144 NaCl, 5.4 KCl, 1.0 MgCl2, 5.0 HEPES, 11.1 Glucose, 0.3 NaH2PO4·2H20, 1.8 CaCl2, 0.1 niflumic acid, and 5.0 4-amino pyridine at 20–21 °C. Tetrodotoxin (TTX, Sigma 5 µmol/L) was included in the perfusate to suppress the inward Na+ current. Voltage clamp was achieved using whole cell ruptured patch technique using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA, USA) operated in switch clamp mode. Pipette resistance was 7–10 M
. The pipette solution contained (mmol/L): 20 KCl, 100 K aspartate, 20 tetraethylammonium chloride, 10 HEPES, 4.5 MgCl2, calculated free [Mg2+] 0.9 mmol/L; 4 Na2ATP, 1 Na2 Creatine phosphate (free Na+=10 mM), 0.1 EGTA, pH 7.25 with KOH. No correction for liquid-junction potentials was applied, the small DC offset observed in Krebs–Henseleit solution was nulled prior to patching on to the cell. Cytosolic loading of Fura-2 was achieved by incubating cardiomyocytes with 5 µmol/L Fura-2-AM at room temperature for 12 min. Cardiomyocytes were voltage clamped at –80 mV and the voltage stepped to –40 mV (50 ms) to inactivate the remaining inward Na+ current, stepping to 0 mV (150 ms) before returning to –80 mV.
2.3 E–C coupling studies at a range of SR Ca2+ loads
The relationship between SR Ca2+ content and Ca2+ transient amplitude was investigated by superfusing cardiomyocytes for set periods of time with thapsigargin (5 µmol/L). This achieved a decrease in Ca2+ transient amplitude and SR Ca2+ content as a result of progressive SERCA2a inhibition. Complete inhibition of the SR was achieved after 100 s perfusion with thapsigargin; rapidly switching to 10 mmol/L caffeine did not generate a Ca2+-release or INCX. Shorter periods of thapsigargin containing perfusion medium achieved intermediate caffeine responses representing intermediate SR Ca2+ contents. Separate groups of cells were exposed to 40 s and 80 s periods of perfusion with thapsigargin, Ca2+ transient amplitude was measured from the last 4 transients before caffeine application. SR Ca2+ content was increased by using a voltage-clamp protocol where a holding potential of –50 mV was used. Measurements of L-type Ca2+ channel current amplitude and the calculation of the integral of this current was used to verify that none of the above protocols caused a significant change in either of these parameters.
2.4 Ca2+ spark and wave measurements in permeabilised cardiomyocytes
Isolated rabbit cardiomyocytes were superfused with a mock intracellular solution and permeabilised using β-escin (Sigma) as detailed previously [6]. 10 µmol/L Fluo-3 was excited at 488 nm (Kr laser) and measured >515 nm using epifluorescence optics of an inverted microscope with a 60x/1.2 NA water-immersion objective lens. Fluorescence was acquired in line-scan mode at 2 ms/line; pixel dimension was 0.29 µm (512 pixels/scan; zoom=1.4). The scanning laser line was oriented parallel with the long axis of the cell and placed approximately equidistant between the outer edge of the cell and the nucleus/nuclei, to ensure the nuclear area was not included in the scan line. To enable this trace to be converted to [Ca2+] a series of calibration solutions were used at the end of each Ca2+ spark measurement period incorporating 10 mmol/L EGTA as previously described [8]. In all experiments concerning Ca2+ sparks, the [Ca2+] in the test solution was 145–160 nmol/L. Ca2+ sparks recorded in Fluo-3 containing solutions were quantified using an automatic detection and measurement algorithm adapted from a previously published method [8]. All Ca2+ spark and wave measurements were made within 2–3 min of cell permeabilisation.
2.5 FK-506 binding protein (FKBP12.6) over-expression within rabbit cardiomyocytes
Recombinant adenoviruses were generated as previously described using cDNA encoding the full open reading frame of the human FKBP12.6 gene [9]. Two populations of adenovirus transfected cardiomyocytes were produced (i) over-expressing FKBP12.6 (Ad-FKBP12.6) and (ii) expressing β-galactosidase as control (Ad-LacZ). Infected cardiomyocytes were washed and subsequently cultured in supplemented M199 medium (Sigma) for 24 h. Verification of transgene expression and virus transfection efficiency has been detailed elsewhere [9]. Previous measurements suggest that the level of FKBP12.6 over-expression was approximately 6-fold that of control cells [9].
2.6 Measurements of SERCA and RyR2 characteristics
Cardiomyocytes in suspension (2x105 cells/ml) were permeabilised by 0.1 mg/ml β-escin (Sigma), placed in a cuvette and stirred to maintain them in suspension (20–22 °C). Fura-2 (10 µmol/L, Molecular Probes) was used to monitor the [Ca2+] within the cuvette using a dual-wavelength spectrophotometer (Cairn Research Ltd). Oxalate (10 mmol/L Sigma) was added to maintain low and constant levels of intra-SR [Ca2+]. This preparation was used to assess SERCA and RyR2 activities as described in Results.
2.7 Statistics
Data were expressed as mean±SEM. For ionic currents, intracellular [Ca2+] and Ca2+ spark variables, comparisons were performed by using the paired Students' t-test and differences were considered significant when P<0.05. ANOVA statistics with a Tukey post-test were used in cases of multiple comparisons.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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3.1 Measurements of intracellular [Ca2+] in voltage-clamped rabbit cardiomyocytes
In paired measurements, the Ca2+ transient amplitude fell to 83±7% (n=13, P<0.05) of control when perfused with 1 µmol/L K201 (Fig. 1A(iii) and C(i)). The decay of the transient was fitted to a mono exponential. K201 (1 µmol/L) caused a small but significant decrease in the rate constant for decay (2.29±0.17 s–1 vs. 2.17±0.15 s–1; control vs. K201, P<0.01). When perfused with 3 µmol/L K201 Ca2+ transient amplitude was reduced to 60±7% (n=6, P<0.05) of control (Fig. 1A(iii) and C(i)). As illustrated in Fig. 1A(i), ICa,L amplitude was monitored by incorporating a pre-pulse to –40 mV for 50 ms to inactivate the remaining inward INa (in the presence of 5 µmol/L TTX). ICa,L was not significantly different from control during perfusion with 1 µmol/L K201 97±7% (n=6), but perfusion with 3 µmol/L K201 resulted in reduction of ICa,L to 66±8.2% (n=7, P<0.05) of the control value (Fig. 1A(ii) and 1C(ii)).
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3.2 SR Ca2+ content as assessed by rapid application of caffeine
When perfused with 1 µmol/L K201, the amplitude of the caffeine-induced Ca2+ release was not significantly different (90±7%, n=10) from control (Fig. 1B and D(i)). However, perfusion with 3 µmol/L K201 reduced the amplitude of the caffeine-induced Ca2+ transient to 74±8.9% of control (P<0.05, n=7; Fig. 1B and D(i)). As described previously [10], the time integral of the INCX can be used as a measure of the amount of Ca2+ extruded by NCX during a caffeine application (an indicator of the SR Ca2+ content). The mean integral of INCX in the cells perfused with 1 µmol/L K201 was not significantly different from control (104±10%, n=4 cells). But in cells perfused with 3 µmol/L (Fig. 1D(ii)), INCX integral was reduced to 65±6.6% (n=5, P<0.05) of control. This supports the conclusion that SR Ca2+ content was significantly reduced by 3 µmol/L but not 1 µmol/L K201.
3.3 Sarcolemmal Ca2+ efflux rates in rabbit cardiomyocytes
Sarcolemmal flux rates can be estimated from the time course of the decay of [Ca2+] after rapid application of 10 mmol/L caffeine. As shown in Fig. 1B and D(i), [Ca2+] decayed with a similar rate constant in cells perfused with both 1 µmol/L (101±3%, n=8 of control) and 3 µmol/L K201 (100±6%, n=7 of control) compared with control. This suggests that the rate of extrusion of Ca2+ via NCX was not affected by K201.
3.4 The gain of E–C coupling
To determine the relationship between SR Ca2+-content and Ca2+ transient amplitude, measurements were made at a range of SR loads. As shown in Fig. 1E, the plot of INCX integral (SR Ca2+ content) and Ca2+ transient amplitude for the control group generated an approximately hyperbolic relationship. Analysis of ICa,L indicated that there were no significant changes in the amplitude or time course of this current in the control and 1 µM K201 data-sets. Therefore, the hyperbolic relationship described by the control data represents the relationship between SR Ca2+ content and the ability of ICa,L to trigger Ca2+ release from the SR; i.e. E–C coupling ‘gain’ [10,11]. The data from the measurements made with 1 µM K201 lies on a gain curve to the right of the control group. K201 appears to decrease Ca2+ transient amplitude by decreasing the fractional release of Ca2+ from the SR.
For comparison, agents known to modulate Ca2+ induced Ca2+ release (CICR) from the SR were studied. Caffeine (0.5 mmol/L) and tetracaine (100 µmol/L) did not change the steady-state Ca2+ transient amplitude compared to control, but significantly altered the SR Ca2+ content. This result contrasts with the effects of K201, where decreased fractional SR Ca2+ release manifests as a decrease in the Ca2+ transient with no detectable changes in SR Ca2+ content. The data for 3 µM K201 was plotted as a single point in Fig. 1E. The data lies to the right of the 1 µM K201 data suggesting reduced E–C coupling gain. However, direct comparisons cannot be made since this effect is accompanied by a reduction in ICa,L.
3.5 Effects of K201 in intact cells during SR-mediated Ca2+ overload
Voltage-clamped cardiomyocytes stimulated at 0.1 Hz were perfused at an extracellular [Ca2+] of 8 mmol/L. The [Na+] of the pipette solution was increased to 40 mmol/L. This consistently resulted in sufficient cellular Ca2+ overload to generate regular, spontaneous Ca2+ release from the SR during the diastolic period. Spontaneous Ca2+ release frequency was monitored for a 50 s period before and after application of 1 µmol/L K201 (Fig. 2A(ii)). Control experiments were also performed with the use of the vehicle (DMSO, Fig. 2A(i)). Ca2+ wave frequency was significantly reduced during application of 1 µmol/L K201 (Fig. 2A(ii) and B(i)) to 61.2±9.67% (n=5; P<0.05) of the pre-K201 control period. The mean Ca2+ transient amplitude over this 50 s period was maintained during the application of 1 µmol/L K201 despite this reduction Ca2+ wave frequency (Fig. 2B(ii)).
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3.6 Effects of K201 in permeabilised cells during SR-mediated Ca2+ overload
Confocal microscopy was used to determine Ca2+ wave characteristics in permeabilised ventricular cardiomyocytes perfused with a mock intracellular solution with 10 µmol/L Fluo-5F (free acid), 350 nmol/L [Ca2+] [7]. Acute permeabilisation permitted functional bypass of sarcolemmal fluxes. The addition of 1 µmol/L K201 for 90 s significantly decreased Ca2+ wave frequency to 77±3.9%, (n=6; P<0.05) of control value (Fig. 3A(i) and C(i)), while 3 µmol/L (n=7) abolished Ca2+ waves (Fig. 3B(i) and C(i)). 1 µmol/L K201 significantly reduced mean Ca2+ wave velocity (to 87±4.2%, n=5; P<0.05, of control) while 3 µmol/L K201 reduced the velocity of the last wave (to 67.7±6.5%, n=6, P<0.05, Fig. 3A(ii), B(ii) and C(ii)). Although these changes in velocity and frequency were observed with 1 µmol/L K201, no change was observed in the mean Ca2+ wave minimum or maximum [Ca2+] (Fig. 3D(i) and (ii)). Neither the peak of the caffeine-induced Ca2+ release during application of 1 µmol K201 nor the amplitude of the response was significantly altered when compared to control (Fig. 3D). In 3 µmol K201, the amplitude of caffeine-induced Ca2+ release was reduced compared to control, this contrasts to the substantial increase in the amplitude of caffeine-induced release observed in 100 µM tetracaine (Fig. 3D(iii) TET).
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3.7 Are the effects of K201 due to alteration in RyR2 Ca2+ sensitivity?
The effects of K201 were compared with 100 µmol/L tetracaine, a drug known to reduce the Ca2+ sensitivity of RyR2 without significant effects on SERCA [12]. As shown in Fig. 3E(i) and (ii), tetracaine abolishes Ca2+ waves in a similar fashion to that observed with 3 µmol/L K201 (Fig. 3B(i)). As reported [10], the peak caffeine-induced Ca2+ release (an indication of the SR Ca2+ content) in tetracaine was higher than control (Fig. 3E(i)). This aspect of the response is different from the effects of K201; 3 µmol/L K201 caused a significant decrease in the amplitude of the caffeine-induced Ca2+ release (Fig. 3D(iii)). A decreased amplitude resulted from an increased minimum [Ca2+] with no change in peak [Ca2+] reached during application of caffeine. This effect was difficult to interpret since the caffeine-induced Ca2+ releases were initiated from different minimum [Ca2+] levels in control and during K201 application. To overcome this issue, the experiments were repeated using a higher concentration of EGTA (150 µmol/L) to abolish Ca2+ waves but not caffeine-induced Ca2+ release (Fig. 3F(i)). Under these conditions, the caffeine-induced Ca2+ release in 1 µmol/L K201 was not significantly different, in 3 µmol/L K201 was 73±10% of control (n=9; P<0.05). 100 µmol/L tetracaine resulted in an increase of Ca2+ release (to 307±17%) (Fig. 3F). This confirms the earlier observation that 3 µmol/L but not 1 µmol/L K201 reduces the SR Ca2+ content.
3.8 The use of local application of caffeine in order to evoke Ca2+ waves in 3 µmol/L K201
Potentially, 3 µmol/L K201 may abolish Ca2+ waves by inhibiting the ability of local release to propagate throughout the cardiomyocyte. To test this hypothesis, caffeine (30 mmol/L) was applied at one end of the cardiomyocyte (Fig. 4A) every 15 s to generate a series of local Ca2+ releases. Under control conditions, each local Ca2+ release propagates rapidly to the other end of the cardiomyocyte (Fig. 4B(i)). Fluorescence signals from a 10 pixel band close to the area of local application of caffeine (L2) and at some distance from the point of initiation (L1) are shown in Fig. 4C(i). These signals are shown during application of 3 µmol/L K201. Prior to the addition of K201, every evoked Ca2+ wave in the control situation propagates to the other end of the cell (Fig. 3C(ii)a). When similar procedures were performed during application of 3 µmol/L K201, local application of caffeine evoked a Ca2+ release at adjacent sites, but only at intervals of 1 min and with a considerably lower propagation velocity (Fig. 4C(ii)b). Attempts to initiate a propagating release at shorter intervals failed (Fig. 4C(ii)c).
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3.9 The effect of 1 µmol/L K201 on Ca2+ sparks
Ca2+ sparks were examined in permeabilised cells perfused with 150–160 nmol/L [Ca2+] and Fluo-3. The line-scan images obtained with and without 1 µmol/L K201 (Fig. 5A(i) and (ii) and mean data (Fig. 5B) show that during application of 1 µmol/L K201 calcium spark duration (iii) and frequency (iv) were decreased to 82% and 63% of control respectively.
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3.10 The effects of K201 on SERCA activity
Measurements from the last Ca2+ wave to occur in 3 µmol/L K201 showed a rate of decay that was significantly slower than control (Fig. 6A(i)) as was the case at 1 µmol/L K201 (Fig. 6A(ii)). This suggests that the rate of Ca2+ uptake by the SR was reduced.
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In order to examine whether SERCA activity was affected by K201, oxalate-supported Ca2+ uptake was measured in aggregates of permeabilised cardiomyocytes in the presence of 7 µmol/L ruthenium red. The rate of uptake of [Ca2+] in the cuvette after addition of an aliquot of CaCl2 was measured both before and after addition of K201. A quantitative description of SERCA activity was obtained by calculating the changes of total [Ca2+] based on the known Ca2+-buffering capacity of the extracellular solution (Fig. 6B(i)). Differentiation of the total [Ca2+] signal yields the rate of Ca2+ uptake that can be plotted against the associated free [Ca2+] (Fig. 6B(ii)). This relationship was fitted with a logistic curve to estimate the free [Ca2+] that generated the half-maximal Ca2+ uptake rate (Km) and the value of Vmax. The mean data show that 1 and 3 µmol/L K201 increased the Km to 107±2% (n=6; P<0.05) and 119±6% (n=7; P<0.05) respectively. The Vmax of SERCA was decreased at 1 and 3 µmol/L K201 to 88±3% (n=6; P<0.05) and 77±3% respectively (Fig. 5D(i) and (ii)).
3.11 The effects of K201 on RyR2 mediated Ca2+ leak
RyR2-mediated Ca2+ efflux in the absence of SERCA activity was observed in β-escin-permeabilised cardiomyocytes after Ca2+ loading the SR. As shown in Fig. 6C(i), the addition of 25 µmol/L thapsigargin and 20 mmol/L caffeine caused a rise of free [Ca2+] within the cuvette as a result of Ca2+ loss from the SR. The rate of Ca2+ rise was substantially reduced by the prior addition of 7 µmol/L ruthenium red to block RyR2 (Fig. 6C(ii)). The rate of rise of Ca2+ was monitored in the absence and presence of 1 µmol/L and 3 µmol/L K201. As shown in Fig. 6C(ii), K201 reduced the rate of Ca2+ leak from the SR. The rate of rise of [Ca2+] was measured at 500 nmol/L, as shown in Fig. 6D (iii), the rate of Ca2+ leak from the SR was reduced in a dose-dependent manner; 1 µmol/L K201 reduced the leak rate to 84±5%, (n=9, P<0.05); 3 µmol/L reduced the rate to 71±6%, (n=9, P<0.05). Ruthenium red (7 µmol/L) reduced the rate of Ca2+ to 12±2% (n=7, P<0.01) of control. K201 had no effect on the residual Ca2+ leak from the SR in the presence of ruthenium red (data not shown). These data support the contention that K201 inhibits RyR2 mediated Ca2+ efflux from the SR.
3.12 The effect of K201 after FKBP12.6 over-expression
FKBP over-expression was used to increase substantially the fraction of RyR bound to FKBP12.6 (Fig. 7). Rabbit cardiomyocytes were permeabilised after 24 h of quiescent culture after transfection with a control virus (Ad-LacZ) or Ad-FKBP12.6. As shown in Fig. 7A and B, perfusion with 400 nmol/L Ca2+ caused regular Ca2+ waves as indicated from the average Fluo-3 fluorescence from a 20 pixel segment of a line-scan confocal image. 3 µmol/L K201 abruptly inhibits Ca2+ waves as previously observed in freshly dissociated cells (Fig. 3). On FKBP12.6 over-expression, the frequency of Ca2+ waves within the permeabilised cardiomyocyte was significantly lower than control, but K201 caused an inhibition of waves with a similar time course to control (Fig. 3B and C). This data indicates that FKBP12.6 over-expression does not decrease the sensitivity of the SR to the inhibitory action of K201.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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This is the first study comparing the effects of K201 on EC coupling in normal and Ca2+ overloaded ventricular cardiomyocytes.
4.1 The effects of K201 on intact cardiomyocytes
Application of 1 µmol/L K201 resulted in a reduction in the amplitude of Ca2+ transients with no change in SR Ca2+ content or ICa,L amplitude. This suggests that ICa,L is less effective at evoking Ca2+ release from the SR, i.e. K201 reduced the gain of EC-coupling thereby reducing the fraction of Ca2+ recirculated through the SR. One possible mechanism for this effect is reduction of the Ca2+ sensitivity of RyR2. However, as previous studies have shown, selective reduction of the Ca2+ sensitivity of RyR2 increases the SR Ca2+ content but does not affect the steady-state Ca2+ transient amplitude [10]. Therefore if K201 acts on RyR2, it must also act in parallel to reduce the SR Ca2+ content, an action consistent with the inhibition of SERCA observed. All of the effects of K201 were reversible, normal parameters were restored after 2–3 min after return to control solution (see one-line supplement).
When normal cardiomyocytes were exposed to a high intracellular [Ca2+], significant spontaneous diastolic Ca2+ release occurred between the stimulated Ca2+ transients. Application of 1 µmol/L K201 resulted in a reduction in the frequency of spontaneous Ca2+ release, similar to that recently reported in unstimulated rat cardiomyocytes [13]. Under these conditions Ca2+ transient amplitude was not reduced by K201. This may be due to a tendency to reduce SR Ca2+ content as a result of SERCA inhibition [11] which is approximately balanced by a tendency to enhanced SR content due to inhibition of spontaneous SR Ca2+ release [14].
4.2 The effects of K201 on SR function in permeabilised cardiomyocytes
Previous studies have shown that reducing the sensitivity of RyR2 to Ca2+ using the drug tetracaine results in an increase in SR Ca2+ content [15]. This was confirmed in the present study. 100 µmol/L tetracaine abolished Ca2+ waves with a similar time course to 3 µmol/L K201 and SR content was increased. But neither 1 nor 3 µmol/L K201 increased SR Ca2+ content. Measurements in higher EGTA concentrations indicated that 3 µmol/L K201 reduced the SR Ca2+ content to 72% of control. These data indicate a more complex mechanism for the action of K201 other than a reduced Ca2+ sensitivity of RyR2 alone.
The rate of decline of the last Ca2+ wave in both 1 and 3 µmol/L K201 was reduced suggesting that SERCA activity is directly affected by K201. This was confirmed by assessment of SERCA activity in oxalate-equilibrated permeabilised cardiomyocytes. A previous study using SR vesicles failed to observe an effect of 1 µmol/L K201 on SERCA-mediated uptake [16]. The reason for this discrepancy is unknown, but the effect observed in this study was small (10%) and therefore may not have been detectable in SR vesicle preparations. Higher K201 concentrations (e.g. 3 µmol/L) caused a further inhibition (20%), but were not investigated in previous studies. RyR2-mediated Ca2+ leak in oxalate-equilibrated cells was reduced by both 1 and 3 µmol/L K201 (Fig. 7C). Together, these results show that K201 has an inhibitory effect on both RyR2 and SERCA activity.
4.3 The effects of K201 on Ca2+ sparks in permeabilised cardiomyocytes
Permeabilised cardiomyocytes were used to examine SR function directly. Under these circumstances, single cardiomyocytes can be superfused with a standardized [Ca2+], [Mg2+], pH, ATP and creatine phosphate concentration. 1 µmol/L K201 significantly reduced the frequency and amplitude of Ca2+ spark characteristics, an effect consistent with actions on both RyR2 and SERCA [12].
4.4 The effects of K201 on Ca2+ waves in permeabilised cardiomyocytes
1 µmol/L K201 significantly reduced the frequency and velocity of Ca2+ waves despite no significant change in SR Ca2+ content. Reduced velocity and frequency is consistent with a reduced Ca2+ sensitivity of RyR2 [17]. Moreover, this effect cannot be attributable to SERCA inhibition since a small degree of inhibition would be expected to have limited effects on Ca2+ wave characteristics [18,19]. Spontaneous Ca2+ waves were abolished with 3 µmol/L K201 in the steady state, but local Ca2+ release elicited by caffeine resulted in a propagated Ca2+ wave at reduced frequency and velocity compared with control. These effects also cannot be explained by inhibition of SERCA alone since this would be expected to reduced Ca2+ wave frequency not abolish waves completely [18,20]. Since Ca2+ sparks are still evident in 3 µmol/L K201, the absence of Ca2+ waves could therefore be due to reduced sensitivity of the RyR2 preventing Ca2+ release propagating throughout the cell. The larger Ca2+ release caused by local application of caffeine is sufficient to trigger a propagated Ca2+ wave. The increased time required between release events suggests that a longer time is required to re-establish the SR Ca2+ content sufficient for a wave.
4.5 Are the RyR2 mediated effects due to alteration of FKBP12.6 binding?
Whilst the effects of SERCA by K201 reported in this study are novel, the effects of K201 on RyR2 in isolated SR vesicle and lipid bilayer experiments have been reported previously [2]. A recent report has shown a similar effect of K201 on isoprenaline-induced transient inward currents (iti) and spontaneous Ca2+ transients in mouse hearts with transgenically reduced FKBP12.6 expression [21]. Furthermore, K201 was ineffective on hearts from FKBP12.6 knockout mice. Taken together the data indicated that K201 acts by restoring FKBP12.6/RyR2 interaction thereby reducing the sensitivity of RyR2 to Ca2+. For this explanation to apply to the current study, the stoichiometry of FKBP12.6 to RyR2 would have to be less than maximal in isolated rabbit cardiomyocytes. To increase the FKBP12.6:RyR2 stoichiometry, FKBP12.6 concentration in the cell was increased by 5–6 fold using an adenoviral transfection system [8]. This has been shown to alter the characteristics of both Ca2+ waves and sparks [8]. Despite this level of FKBP12.6, 3 µmol/L K201 was still able to suppress Ca2+ waves to a similar degree to control cardiomyocytes while 1 µmol/L K201 had similar effects in all experimental groups (data not shown). The similarities of the response of K201, despite markedly different levels of FKBP12.6, suggest that the sensitivity of RyR2 to K201 is unaffected by increases in cytoplasmic FKBP12.6 above control levels.
In summary, this study has shown that 1 µmol/L K201 inhibited both RyR2 and SERCA activity without affecting sarcolemmal Ca2+ influx or efflux via the L-type Ca2+ channel or NCX activity respectively. Abnormal diastolic Ca2+ leak, in the form of spontaneous Ca2+ waves, was suppressed without alteration of SR Ca2+ content. Evidence of the inhibition of Ca2+ influx (via the L-type Ca2+ channel) and a reduction in SR Ca2+ content was only evident at a higher concentration of K201 (3 µmol/L) and was accompanied by a more profound inhibition of SERCA- and RyR2-mediated Ca2+ flux. It is therefore possible that the cellular basis for the anti-arrhythmic effect of K201 demonstrated in previous studies is via the drug's ability to affect multiple components of EC-coupling.
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Appendix A. Supplementary data |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionAppendix A. Supplementary dataReferences |
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.06.014.
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Acknowledgements |
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We would like to thank Aileen Rankin and Anne Ward for preparation of cardiomyocytes and virus transfection. This work was funded by the British Heart Foundation, Medical Research Scotland, Tenovus Scotland, The Royal Society and the Deutsche Forschungsgemeinschaft DFG HA 1233/7-3 (TS). The authors thank Aetas Pharma Ltd. for the gift of K201.
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