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Cardiovascular Research 2005 67(4):667-677; doi:10.1016/j.cardiores.2005.04.023
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Copyright © 2005, European Society of Cardiology

Effects of calsequestrin over-expression on excitation–contraction coupling in isolated rabbit cardiomyocytes

Stewart L.W. Millera, Susan Curriea, Christopher M. Loughreya, Sarah Kettlewella, Tim Seidlerb, Deborah F. Reynoldsa, Gerd Hasenfussb and Godfrey L. Smitha,*

aInstitute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
bDepartment of Cardiology and Pneumology, Georg-August-University Goettingen, D-37075 Goettingen, Germany

* Corresponding author. Tel.: +44 141 3305963; fax: +44 141 3304612. Email address: g.smith{at}bio.gla.ac.uk

Received 15 December 2004; revised 19 April 2005; accepted 20 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study investigated the role of calsequestrin (CSQ) in the control of excitation–contraction (E–C) coupling in the heart.

Methods: CSQ over-expression was induced in isolated rabbit ventricular cardiomyocytes using an adenovirus coding for rabbit CSQ (Ad-CSQ). After 24 h of culture, CSQ protein expression was increased by 58 ± 18% (n = 10). An adenovirus coding for β-galactosidase (Ad-LacZ) was used as a control.

Results: In voltage-clamped, Fura-2-loaded cardiomyocytes, L-type Ca2+ current (ICa,L) and Ca2+ transient amplitude were both increased in the Ad-CSQ group by ~78%. Doubling the external Ca2+ concentration in the control group (Ad-LacZ) increased the LTCC amplitude to a similar degree (85 ± 6%), but increased the Ca2+ transient amplitude by 149 ± 13%. This suggests that SR Ca2+ release may be inhibited upon CSQ over-expression. Alternatively, nifedipine (0.5 µM) was used to reduce ICa,L in Ad-CSQ-transfected cells to values comparable to control (Ad-LacZ). Under these conditions, Ca2+ transient amplitude was not different from Ad-LacZ, but the SR Ca2+ content was ~60% higher as assessed by both the caffeine-induced Ca2+ release and the accompanying Na+/Ca2+ exchanger current (INCX). The cause of the increased ICa,L is unknown. No change in the expression level of the {alpha}1-subunit of the L-type Ca channel was observed. β-Escin-permeabilized cardiomyocytes were used to study Ca2+ sparks imaged with Fluo-3 at 145–155 nmol/L [Ca2+]. Spontaneous Ca2+ spark frequency, duration, width, and amplitude were unchanged in the Ad-CSQ group, but SR Ca2+ content was 48% higher than Ad-LacZ.

Conclusions: CSQ over-expression increased SR Ca2+ content but reduced the gain of E–C coupling in rabbit cardiomyocytes.

KEYWORDS E–C coupling; SR (function); Calcium (cellular); Ion channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The process of excitation–contraction (E–C) coupling in cardiomyocytes involves depolarisation of the sarcolemma and the opening of voltage activated L-type Ca2+-channels (LTCC). Ca2+ influx via these channels stimulates further Ca2+ release from the sarcoplasmic reticulum (SR) via clusters of Ca2+ release channels (ryanodine receptors, RyRs). SR Ca2+ release is terminated by closure of RyR, the mechanism underlying this process is uncertain [1]. Factors intrinsic to the RyR cluster have been proposed [2]. Alternatively closure may be controlled by cytosolic modulators of RyR [3], or by low affinity Ca2+ sensors within the SR lumen [4]. Luminal [Ca2+] is buffered by calsequestrin (CSQ) [5]. This protein may also act as a low affinity sensor of luminal [Ca2+] to modulate RyR activity via the protein's junctin and triadin [6–8]. Exogenous application of CSQ decreased the open probability of isolated RyRs from skeletal [7] and cardiac muscle [8]. Transgenic mice over-expressing CSQ (by a factor of 10 or more) showed a significantly higher SR Ca2+ content, but intracellular Ca2+ transients were dramatically reduced [9–11]. This supports the view that CSQ may have an inhibitory effect on RyR activity, but concomitant changes in expression of other proteins made it difficult to attribute the changes solely to CSQ. Recent work using acute over-expression (by a factor of ~4 x over 48–56 h) observed an increased SR Ca2+ release and prolongation of the release phase [12]. This suggests that the buffer action of CSQ delays the inactivation of RyR, but no inhibitory effect on RyR was evident. In the present study, adenovirus mediated over-expression of CSQ was used to generate a limited over-expression of CSQ (~1.6 x over 24 h) in rabbit cardiomyocytes. An increased SR Ca2+ content and a reduction of E–C coupling gain were observed.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Adenoviral over-expression of CSQ in isolated adult rabbit cardiomyocytes
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). The rabbit cardiac CSQ gene (Genbank# X55040 [GenBank] ) containing the full-length cDNA in pCI-neo was cloned and ligated downstream of an immediate early CMV promoter into vector pACCMV using primers creating KpnI and HindIII sites. Recombination with pJM17 plasmid and production of replication deficient adenovirus was carried out according to standard procedures [13]. Isolation and culture of adult rabbit cardiomyocytes was carried out as described previously [14,15]. Adenoviral infection with a multiplicity of infection (MOI) of 100 was performed to produce two populations of adenovirus transfected cardiomyocytes (i) over-expressing CSQ (Ad-CSQ) and (ii) expressing β-galactosidase as control (Ad-LacZ). Infected cardiomyocytes were subsequently cultured in supplemented M199 medium (Sigma) for 24 h.

2.2. Measurements of protein expression levels
Immunoblotting used the NuPAGE system (Invitrogen) with 10% Bis–Tris gels and MOPS buffer. CSQ in cell lysates from Ad-LacZ and Ad-CSQ transfected cells was detected with mouse monoclonal anti-CSQ at 1:200 dilution (gift from Prof. J.M. East, University of Southampton). CSQ protein was normalised to a standard Ad-LacZ transfected lysate preparation and immunoblots quantified using image analysis software (Quantity One, BioRad). Immunoblots for the {alpha}1-subunit of the LTCC and plasma-membrane Ca2+-ATPase (PMCA) were done under similar conditions. RyR2 expression was measured using a maximal 3H ryanodine binding assay. Separate sarcolemmal (SL)-enriched and SR-enriched membrane fractions of cardiac tissue were prepared using a similar protocol to that described previously [16].

2.3. Voltage clamp and intracellular [Ca2+] measurements in rabbit cardiomyocytes
After 24 h incubation with the virus, isolated cardiomyocytes were superfused with a HEPES-based Tyrode's solution at 20–21 °C in a chamber mounted on the stage of an inverted microscope. Voltage clamp was achieved using whole cell ruptured patch technique using an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA, USA) in discontinuous (switch clamp) mode. Pipette resistance was 7–10 M{Omega}. [Ca2+]i was measured from Fura-2 fluorescence signals at 100 Hz using a dual wavelength spectrophotometric method described previously [17]. Cytosolic loading of Fura-2 was achieved by incubating cardiomyocytes with 5 µmol/L Fura-2-AM at room temperature for 12 min.

2.4. Electrophysiology protocols
2.4.1. E–C coupling protocol
Isolated rabbit cardiomyocytes were held at –80 mV and the voltage stepped to –40 mV for 50 ms in the presence of 5 µmol/L TTX to inactivate the remaining inward Na+ current, before stepping to 0 mV for 150 ms. This protocol was repeated at 0.5 Hz for 80 s to achieve steady-state Ca2+ transients. SR Ca2+ content and NCX activity were then estimated by rapidly switching to a solution containing 10 mmol/L caffeine and 10 mmol/L BDM to cause SR Ca2+ release 1–1.5 s after the end of the last voltage-clamp protocol. Preliminary studies indicated that caffeine-induced cell shortening frequently caused a dramatic reduction in the microelectrode seal resistance in 1 day cultured cells. This was prevented by the inclusion of BDM in the caffeine solution. In the continued presence of caffeine/BDM the SR is unable to re-accumulate Ca2+ and elimination of Ca2+ is mainly due to NCX. A correction was made for non-NCX Ca2+ removal mechanisms estimated from the Ca2+ decay obtained by rapidly switching to caffeine/BDM in the presence of 10 mmol/L NiCl2 [18].

2.4.2. Manipulation of SR Ca2+ load–(i) increased SR Ca2+ load
Three methods were used, Firstly, 20 mmol/L citrate was added to the pipette solution [4] to increase SR buffer capacity in Ad-LacZ transfected cardiomyocytes. Secondly, periods of 1 Hz stimulation (1–2 min) were used to increase SR content without affecting ICa,L or other sarcolemmal flux pathways. Thirdly, the holding potential was maintained at –60 mV, thus reducing Ca2+ efflux via NCX activity, raising diastolic [Ca2+] and therefore SR content (ii) decreased SR Ca2+ load: 5 µmol/L thapsigargin was washed on to AdLacZ transfected cardiomyocytes contracting in the steady state to reduce SR Ca2+ load.

2.4.3. ICa,L measurements
From a holding voltage of –80 mV, the membrane voltage (Em) was stepped to –40 mV for 50 ms in the presence of 5 µmol/L TTX to inactivate the remaining inward Na+ current. Thereafter, Em was held at a range of voltages (–40 to +80 mV) for 150 ms. In each cardiomyocyte, the protocol was repeated after perfusion with 0.3 mM Cd2+ to inhibit ICa,L and the difference currents calculated.

2.4.4. INCX measurements
After achieving the whole-cell configuration, a period of 4–5 min was allowed for dialysis of the pipette solution into the cell. Membrane current (Im) was measured in response to a 3 s ramp from –120 mV to +80 mV from a holding potential of –80 mV following a previously published protocol [19]. The ramp protocol was performed at 0.1 Hz until steady-state currents were achieved, whereupon data from five ramps was averaged. The protocol was repeated in the presence of 5 mmol/L NiCl2 to obtain the background current, and this was subtracted to obtain the current attributable to NCX (Ni-sensitive current).

2.5. Solutions used in voltage clamp protocols
The Tyrode's solution contained (mmol/L): NaCl (140), KCl (4), HEPES (5), MgCl2 (1), CaCl2 (1.8), glucose (11.1), pH 7.4 with NaOH. This solution was modified as appropriate for each experimental protocol. For Na+–Ca2+ exchanger (NCX) activity studies, the superfusate contained added 4-aminopyridine (5 mmol/L, to block K+ currents) and Niflumic acid (0.1 mmol/L, to block Ca2+-activated Cl– currents). The pipette solution contained (mmol/L): KCl (20), K aspartate (100), tetraethylammonium chloride (TEACl, 20), HEPES (10), MgCl2 (4.5, calculated free Mg2+{approx}0.9 mmol/L), disodium ATP (4), disodium creatine phosphate (1), EGTA (0.01), pH 7.25 with KOH. For ICa,L studies, 5 mmol/L BAPTA was added. For INCX studies the superfusate was also K+-free, KCl was replaced by CsCl, with added strophanthidin (0.01 mmol/L) and nifedipine (0.01 mmol/L). The pipette solution contained (mmol/L): CsCl (45), EGTA/Ca2+ EGTA (Cs+ 100, EGTA 50, Ca2+ 25), HEPES (20), MgCl2 (11, calculated free Mg2+{approx}1.2 mmol/L), Na2ATP (10); pH 7.25. This pipette solution was designed to heavily buffer [Ca2+]i to 252 ± 4 nmol/L and conditions were confirmed in Fura-2-loaded cells (n = 11, data not shown).

2.6. Ca2+ spark measurements in permeabilised cardiomyocytes
Isolated rabbit cardiomyocytes were superfused with a mock intracellular solution and permeabilized by perfusing for 30–45 s with a 0.1 mg/mL β-escin (Sigma). Fluo-3 (10 µmol/L) in the perfusing solution was excited using a confocal microscope (BioRad Radiance 2000) in linescan mode at a rate of 2 ms/scan. In all experiments concerning Ca2+ sparks, the [Ca2+] in the test solution was 145–155 nmol/L. Ca2+-sparks recorded in Fluo-3 containing solutions were quantified using an automated detection and measurement algorithm adapted from a previously published method[20]. All Ca2+ spark measurements were made within 2 min of cell permeabilisation.

2.7. Statistics
Data was expressed as means ± S.E.M. For ion currents and Ca2+ transients, comparisons were performed by using the Anova multivariate analysis and a Tukey post-test. Differences were considered statistically significant was when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of infection with Ad-CSQ on protein expression in rabbit adult cardiomyocytes
Infection of cardiomyocytes with adenovirus encoding for rabbit CSQ caused an increase in CSQ protein expression in a dose dependent manner (Fig. 1A(i)). Quantitative Western blotting indicated that 24 h after infection (Ad-CSQ, 100 MOI) the average CSQ levels had increased to 158 ± 18% (n = 10, P<0.05) when compared to Ad-LacZ controls (Fig. 1A(ii)). This increase in CSQ levels was not accompanied by changes in the LTCC protein expression as assessed by immunoblots for the {alpha}1-subunit (Fig. 1B(i)). The plasmalemmal Ca2+ ATPase (PMCA) expression was not significantly altered (Fig. 1B(ii)), nor was the expression of RyR as assessed by maximal binding of 3H ryanodine (Fig. 1B(iii)). CSQ appeared to be entirely expressed in the SR since no immunoblot signal could be detected in sarcolemmal (SL) membrane fractions, but strong signals were evident in SR membrane fractions as illustrated in Fig. 1C (typical of 4 separate measurements).


Figure 1
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  		Fig. 1 Panel A: verification of CSQ protein over-expression in isolated adult rabbit cardiomyocytes 24 h after transfection with Ad-CSQ virus. Dose dependence of the over-expression is indicated in the Western blot shown in panel A(i), 10 µg total protein was loaded into each. The mean increase of CSQ expression observed at MOI=100, compared to transfection with Ad-LacZ (MOI=100) is shown in A(ii). Panel B summarised the measurements of LTCC, PMCA and RyR expression. Panel C shows the detection of CSQ and NCX in sarcolemmal (SL) and SR membrane fractions using immunoblot techniques. The protein loads used are shown below the sample blots.

 
3.2. Intracellular Ca2+ measurements in voltage-clamped rabbit cardiomyocytes
As illustrated in Fig. 2A, there was a significant increase in peak-systolic [Ca2+] and end-diastolic [Ca2+] in voltage-clamped rabbit cardiomyocytes after Ad-CSQ transfection (Fig. 2C(i) and (ii)). The amplitude of the Ca2+ transient was calculated as the difference between peak [Ca2+] and end diastolic [Ca2+]. The mean amplitude of the Ca2+ transient was also increased by 78% (Ad-LacZ, 117 ± 20 nmol/L n = 17; Ad-CSQ, 209 ± 19 nmol/L, n = 19, P<0.05). In addition to the measurements of ICa,L amplitude, the time-integral of ICa,L was calculated and converted to a Ca2+ influx (normalized to cell capacitance). Both the amplitude of ICa,L and the integral of the current were significantly increased by (~78%) in Ad-CSQ transfected cardiomyocytes (Fig. 2D(i) and (ii)). In a separate set of experiments, LTCC (amplitude and integral) was increased in Ad-LacZ cardiomyocytes to an extent comparable to that seen in the Ad-CSQ group by increasing extracellular [Ca2+] to 3.6 mmol/L (Fig. 2A(iii), Hi-Ca). LTCC amplitude was ~80% larger and the Ca2+ transient amplitude was increased by ~149 ± 13% (n = 12) in the Hi-Ca group. As shown in Fig. 2C and D, mean systolic [Ca2+] was significantly higher in the Hi-Ca group than the Ad-CSQ group yet diastolic [Ca2+] and ICa,L characteristics were comparable.


Figure 2
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  		Fig. 2 Depolarization induced Ca2+ transients recorded from Ad-LacZ and Ad-CSQ transfected cardiomyocytes and Ad-LacZ cardiomyocytes exposed to 3.6 mmol/L extracellular Ca2+ (Hi-Ca). Panel A: records of Em, Im and [Ca2+]i from single cardiomyocytes (average of 10 signals). Panel B shows Im recorded on depolarization from the pre-pulse potential of –40 mV to 0 mV for 150 ms to illustrate the amplitude and time course of the ICa,L (extracts from panel B(i) and (ii). Panel C shows mean ± S.E.M. (n = 10) of: (i) peak systolic [Ca2+]i, (ii) end diastolic [Ca2+]i. Panel D shows mean ± S.E.M. values of: (i) ICa,L amplitude (ii) Ca2+ influx via ICa,L. * indicates significant difference from Ad-LacZ values P<0.05; # indicates significant difference between Ad-CSQ and Hi-Ca groups P<0.05.

 
3.3. SR Ca2+ content–rapid application of caffeine in rabbit cardiomyocytes
Application of caffeine caused a rapid increase of [Ca2+]i as a result of SR Ca2+-release. The subsequent reduction of [Ca2+]i results from extrusion of Ca2+ across the sarcolemma mainly via NCX. The extrusion of Ca2+ via NCX generates a transient inward current, the amplitude and time-course of which was monitored together with the Ca2+-transient (Fig. 3). Caffeine at a concentration of 10 mmol/L was rapidly applied within 1–1.5 s of the end of a train of 40 voltage-clamp pulses to assess a steady-state SR Ca2+ load. The peak of the caffeine-induced Ca2+ release was significantly larger in cardiomyocytes transfected with Ad-CSQ, suggesting an increased SR Ca2+ content (Ad-LacZ, 585 ± 86 nmol/L n = 17; Ad-CSQ, 939 ± 107 nmol/L, n = 19, P<0.05). The peak of the transient inward current was significantly larger in Ad-CSQ transfected cells (Fig. 3B(ii)). The mean integral of the NCX-mediated inward current (INCX) was significantly larger in the CSQ-transfected cardiomyocytes compared to the Ad-LacZ transfected group (normalised to cell capacitance, Fig. 3B(iii)) supporting the conclusion that SR Ca2+ content was increased in CSQ over-expressing cardiomyocytes. The time course of the inward current decay and the corresponding decrease in [Ca2+] are shown in Fig. 3A(i) and (ii). INCX and [Ca2+]i decayed at a similar rate in both cell types. As described in previous publications [18,21,22], these decays were fitted to a single exponential and mean rate constants were calculated (Fig. 3C). The rate constants for [Ca2+] decay (Fig. 3C(i)) and INCX decay (Fig. 3C(ii)) indicate a similar rate of extrusion of Ca2+ in both cell types. This suggests that sarcolemmal Ca2+ extrusion was unaffected by CSQ over-expression. Since INCX is stoichiometrically linked to the total number of Ca2+ ions extruded from the cell, this protocol can be used to assess the intracellular Ca2+ buffer power [18,23]. The INCX from the decay phase of the caffeine-induced current is used to calculate the total Ca2+ extruded from the cell. The change in total cell Ca2+ can be plotted against the [Ca2+]i to generate a buffer relationship (Fig. 3D(i)). The gradient of this relationship reflects the intracellular buffer power, no difference in mean gradient was observed between the two experimental groups (Fig. 3D(ii)) indicating that CSQ over-expression did not alter the cytoplasmic Ca2+ buffer capacity. The data concerning SR Ca2+ content in the Ad-CSQ group was also compared with data from cells exposed to raise extracellular Ca2+ (Hi-Ca). As shown in Fig. 3, under these conditions, caffeine induced Ca2+ release and the integral of INCX (SR Ca2+ content) were increased to values comparable to the Ad-CSQ group. The mean rate constant for the decay of intracellular [Ca2+] and INCX were both significantly reduced in the Hi-Ca group (Fig. 3C). This data and absence of significant changes of the peak INCX is consistent with reduced forward mode activity of NCX in raised extracellular [Ca2+].


Figure 3
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  		Fig. 3 Caffeine-induced SR Ca2+ release and the corresponding membrane currents from three cardiomyocytes of comparable capacitance (Ad-LacZ, 101 µF; Ad-CSQ 98 µF, Hi-Ca 111 µF). Panel A: records of [Ca2+]i and Im recorded on application of 10 mmol/L caffeine in Ad-LacZ and Ad-CSQ groups. Panel B(i), for Ad-LacZ (n = 17) and Ad-CSQ (n = 19) groups: Peak [Ca2+]i; (ii) Peak INCX; (iii) INCX time integral. Panel C: mean ± S.E.M. values: (i) rate constant for the decay of the caffeine induced Ca2+ transient; (ii) rate constant for the recovery of Im in response to caffeine. Panel D, plot of total Ca2+ efflux from the cardiomyocyte (derived from INCX) and free [Ca2+]i. The gradient of this graph reflects the Ca2+ buffer power of the cytosol. The mean gradient of this relationship (corrected for cell capacitance) is shown in (ii). The mean capacitance of the Ad-LacZ group was 91 ± 3 µF (n = 15), the Ad-CSQ 95 ± 4 µF (n = 17). * indicates significant difference from Ad-LacZ values P<0.05; # indicates significant difference between Ad-CSQ and Hi-Ca groups P<0.05.

 
3.4. ICa,L and INCX current–voltage relationships after CSQ over-expression
The current–voltage (I–V) relationship and the voltage sensitivity of inactivation of ICa,L was examined. Intracellular Ca2+-dependent modulation of ICa,L was inhibited by the dialysis of the cardiomyocyte with 5 mmol/L BAPTA. A significantly larger ICa,L was observed after CSQ over-expression with no apparent effect on the voltage dependence of inactivation of the current (Fig. 4A and B). Direct measurement of INCX was performed using a previously established ramp clamp protocol [19,24]. There was no significant change in INCX across the range of voltages studied (–120 to +80 mV) on over-expression of CSQ (Fig. 4B).


Figure 4
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  		Fig. 4 Panel A(i) Voltage clamp protocol used to investigate the I–V relationship of ICa,L. Below the superimposed voltage traces are typical Im records in absence and presence of Cd2+ (0.1 mmol/L). The difference current is attributed to ICa,L. Panel A(ii) shows the average I–V relationship of ICa,L. Panel B(i) Voltage clamp protocol used to investigate voltage-dependent inactivation of the ICa,L. Typical current traces under control conditions and in the presence of Cd2+ are shown. Panel B(ii) shows the average voltage-sensitivity of inactivation of ICa,L measured in Ad-LacZ and Ad-CSQ cells. Solid lines represent the best fit to a the Boltzman equation of the form V = (Imin–Imax)/(1+exp((V–V50)/slope))+Imax. The best-fit parameters to the curves shown in panel B are: Ad-LacZ group, Imax=–1.24 x 10–3 ± 4 x 10–5 nA/µF Imin=4 x 10–5 ± 5 x 10–55 nA/µF, V50=–7.26 ± 1.14 mV slope=5.1 ± 1.0; Ad-CSQ group, Imax=–2.03 x 10–3 ± 7 x 10–55 nA/µF Imin=9 x 10–5 ± 8 x 10–55 nA/µF, V50=–7.3 ± 1.1 mV, slope=5.1 ± 1.0. R2=0.99 for both curves. Panel C(i) Voltage ramps from –120 to +80 mV were used under control conditions and in the presence of 5 mmol/L Ni2+ to generate the I–V relationship for INCX. The difference represents the Ni2+ sensitive current attributed to INCX. Panel C(ii) shows the average I–V relationship.

 
3.5. Manipulation of ICa,L in Ad-CSQ transfected cardiomyocytes
A series of preliminary experiments established that 0.5 µmol/L nifedipine was required to reduce the L-type Ca2+ current amplitude in Ad-CSQ transfected cardiomyocytes to an amplitude similar to that observed in the Ad-LacZ group. As shown in illustration in Fig. 5A, using this concentration of nifedipine, the amplitude of the ICa,L (Fig. 5A(i)) and the rate of inactivation the ICa,L (Fig. 5A(ii)) were comparable in myocytes from each group. The average ICa,L amplitude and integral in both experimental groups were well matched (Fig. 5B(i) and (ii)), and the mean rate constant for the inactivation of ICa,L was not significantly different (Ad-LacZ, 28.0 ± 0.3 s–1 n = 10; Ad-CSQ+Nif, 24.2 ± 0.4 s–1, n = 9). As illustrated in Fig. 5A(iii), under these conditions, both peak systolic [Ca2+] and end diastolic [Ca2+] in the Ad-CSQ group were significantly lower than in Ad-LacZ transfected cardiomyocytes (mean values shown in Fig. 5B(ii) and (iii)), but Ca2+ transient amplitude was not significantly different (117 ± 19 nmol/L vs. 111 ± 20 nmol/L, n = 6). The time course of the Ca2+ transients were also similar, the maximum rate of rise of the transient (Ad-LacZ, 5.0 ± 0.7 nmol/L/ms n = 10; Ad-CSQ, 4.4 ± 0.3 nmol/L/ms, n = 9) and the rate constant for the decay (Ad-LacZ, 1.67 ± 0.08 s–1 n = 10; Ad-CSQ+Nif, 1.01 ± 0.11 s–1, n = 9) were not different. Rapid application of caffeine revealed a significantly increased peak caffeine-induced Ca2+ release and INCX time-integral in Ad-CSQ transfected cardiomyocytes (Fig. 5C, P<0.05). These values in Ad-CSQ cells treated with nifedipine were similar to those observed in the absence of drug (Fig. 3B). Therefore the increased SR Ca2+ content observed in CSQ over-expressing cardiomyocytes was retained after normalisation of the ICa,L to control levels using nifedipine.


Figure 5
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  		Fig. 5 E–C coupling in Ad-CSQ transfected cardiomyocytes in the presence of 0.5 µM nifedipine (Ad-CSQ+Nif). Panel A(i) shows superimposed records of Im resulting from voltage clamp step from –40 mV to 0 mV for 150 ms recorded from individual cardiomyocytes with comparable capacitance from the Ad-LacZ (87 µF) and Ad-CSQ+Nif (83 µF) groups. Panel A(ii) shows the decay phase of the individual currents (normalised). The solid lines through the current records are best-fit exponential curves, the rate constants were: Ad-LacZ, 27.8 ± 0.1 s–1 Ad-CSQ+Nif, 24.0 ± 0.1 s–1. Panel A(iii) Ca2+ transients recorded from the individual cardiomyocytes. Panel B shows mean ± S.E.M. of: (i) ICa,L amplitude (ii) peak systolic [Ca2+]i, (iii) end diastolic [Ca2+]i using the standard E–C coupling voltage clamp protocol (Ad-LacZ n = 6, Ad-CSQ+Nif, n = 8), P<0.05. Panel C, (i) Peak [Ca2+]i and (ii) INCX time integral in response to caffeine (10 mmol/L), Ad-LacZ (n = 6) and Ad-CSQ (n = 8), P<0.01. Panel C, Relationship between INCX integral (an index of SR Ca2+ content) and Ca2+ transient amplitude and ICa,L amplitude for cardiomyocytes from the Ad-LacZ group (Control lhblk, n = 17; 5 µmol/L thapsigargin 40 s exposure, {blacktriangleup}, n = 16; 100 s exposure {diamondsuit}, n = 16; 20 mmol/L Citrate bullet, n = 6; 1 Hz stimulation {blacktriangleright}, n = 6; holding potential of –60 mV {diamond}, n = 10 and Ad-CSQ+Nif group {square}, n = 6; 5 µmol/L thapsigargin 40 s exposure {triangleup}, n = 7; 100 s exposure {diamond}, n = 8; holding potential of –60 mV {triangleleft}, n = 8). Measurements represent steady state values.

 
3.6. Relationship between SR Ca2+-content and Ca2+-transient amplitude in rabbit cardiomyocytes
To determine the relationship between SR Ca2+-content and Ca2+ transient amplitude, measurements were made after experimental manipulations to raise or lower SR Ca2+ content (see Methods). As shown in Fig. 5D, the plot of INCX integral and Ca2+-transient amplitude for the Ad-LacZ group generated an approximately hyperbolic relationship. Analysis of ICa,L indicated that there were no significant changes in the amplitude (Fig. 5D). The hyperbolic relationship described by the Ad-LacZ data represents the relationship between SR Ca2+ content and the ability of a constant ICa,L to trigger Ca2+ release from the SR; i.e. E–C coupling ‘gain’ [23]. The data from the Ad-CSQ+Nif group lies to the right of the relationship described for the Ad-LacZ group. Therefore decreased E–C coupling ‘gain’ accompanies CSQ over-expression.

3.7. Ca2+ sparks and caffeine-induced Ca2+ release in permeabilised cardiomyocytes over-expressing CSQ
An investigation of the direct effects of CSQ over-expression on Ca2+ sparks in intact rabbit cardiomyocytes is complicated by the rapid sarcolemmal extrusion of intracellular Ca2+ via NCX and loss of SR Ca2+ during the quiescent periods required for Ca2+ spark recording. For this reason, sarcolemmal fluxes were functionally by-passed by the acute permeabilization of the sarcolemma with β-escin. Under these circumstances, single cardiomyocytes can be superfused with a standardized [Ca2+] and pH in the presence of ATP and creatine phosphate. Ca2+ spark activity was monitored by the inclusion of 10 µmol/L Fluo-3 in the perfusing solution. A typical record from a single cardiomyocyte is shown in Fig. 6A. SR Ca2+-content was assessed at the end of periods of spark activity by rapid application of 10 mmol/L caffeine. Typical sections of line scan images are shown in Fig. 6B. The results of spark analysis algorithm applied to data from a number of cardiomyocytes are shown in Fig. 6D. It can be seen from this summary that the mean values of peak, width, duration and frequency of the Ca2+ sparks were unaffected by CSQ over-expression compared to Ad-LacZ. The spark peak, duration and width distribution histograms were all of similar shape (results not shown). However, the amplitude of caffeine-induced Ca2+ release was significantly larger in the Ad-CSQ group (Fig. 6C).


Figure 6
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  		Fig. 6 Ca2+-sparks in permeabilized cardiomyocytes transfected with Ad-LacZ (control) and Ad-CSQ. Panel A: intracellular [Ca2+] signal calculated from the mean Fluo-3 fluorescence signal of a 20 pixel intracellular region on perfusion with 150 nmol/L Ca2+ (50 µmol/L EGTA). Arrow indicates the time of application of 10 mmol/L caffeine. Panel B: line scan epifluorescence image from single permeabilized cardiomyocytes 24 h after infection with Ad-LacZ and Ad-CSQ. Panel C mean values of peak [Ca2+] in response to caffeine, *P<0.05. Panel D: mean ± S.E.M. values for: (i) peak F/Fo; (ii) spark frequency; (iii) spark width (iv) spark duration; Results compiled from 927 events from 12 cells (Ad-LacZ) and 1165 events from 17 cells (Ad-CSQ).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study shows that a limited increase in CSQ expression increased SR Ca2+ content but decreased the gain of E–C coupling in isolated rabbit cardiomyocytes.

4.1. Effects of CSQ over-expression on sarcolemmal Ca2+ entry
This study revealed that CSQ over-expression significantly increased ICa,L amplitude without any apparent change in LTCC protein expression. The increased current was not accompanied by any obvious change in the voltage-sensitivity or kinetics of inactivation, therefore it is unlikely that the change of ICa,L amplitude is an indirect consequence of altered Ca2+ transient. Alternatively, increased current may be due to altered sub-unit expression or changes in the phosphorylation status of the LTCC. Previous studies have shown ICa,L to be either reduced, with slower inactivation kinetics [25,11] or unaltered [10,12] on CSQ over-expression. Differences in over-expression methodology, the level of over-expression or the species differences may be the basis for the inconsistencies between studies. The mechanism linking CSQ over-expression to changes in ICa,L is unknown. No CSQ immunoblot signals were observed in a sarcolemmal membrane fraction (Fig. 1D). Therefore the possibility that CSQ over-expression interferes with sarcolemmal processes via the inappropriate insertion of CSQ in the sarcolemma was discounted.

4.2. Effects of CSQ expression on E–C coupling gain
Mimicking the increased ICa,L amplitude observed in the Ad-CSQ group by raising extracellular [Ca2+] increased the Ca2+ transient amplitude to a greater extent than that observed in the Ad-CSQ group (Fig. 2) despite comparable SR Ca2+ content (Fig. 3). This suggests that ICa,L is less effective at releasing Ca2+ from the SR after Ad-CSQ over-expression. Interestingly, raised extracellular [Ca2+] was associated with decreased rate of decay of intracellular Ca2+ and INCX in caffeine. This is to be expected since increased extracellular Ca2+ will reduce the activity of NCX in the forward mode.

Upon normalisation of the ICa,L using nifedipine, the SR Ca2+ content in the Ad-CSQ group remained ~2 x higher than control values while the Ca2+ transient decreased to values comparable to the control (Ad-LacZ) group. This effect of nifedipine is agrees with previous work showing that reduced ICa,L amplitude caused a reduced amplitude of the Ca2+ transient but not the SR Ca2+ content in rat myocytes [26]. The result is consistent with the comparison between the Ad-CSQ and Hi-Ca groups, i.e. ICa,L is less effective at triggering SR Ca2+ release in the Ad-CSQ group. Systolic and diastolic [Ca2+] were significantly lower than control (Ad-LacZ) values. The reason for the lowered diastolic [Ca2+] is not known, but it suggests either a reduced diastolic Ca2+ leak from the SR or enhanced Ca2+ extrusion from the cell during diastole.

The term ‘E–C coupling gain’ represents the effectiveness of the L-type Ca2+ channel to release Ca2+ from the SR. As shown in Fig. 5D, for constant ICa,L amplitude, the ‘gain’ is dependent on the SR Ca2+ content with an approximately hyperbolic relationship. The data points corresponding to the Ad-CSQ+Nif data are clearly to the right of the control relationship, indicating that at all SR Ca2+ loads, the Ca2+ transient was smaller than control. This indicates that CSQ over-expression reduced the sensitivity of the Ca2+-induced Ca2+ release mechanism. This result is consistent with two independent transgenic studies [10,9]. However, CSQ over-expression (~4 x) in isolated rat myocytes increased Ca2+ transient amplitude and caffeine induced release [12]. The ‘gain’ of E–C coupling in this latter study was not thought to be dramatically different since the results were mimicked by using citrate to boost SR Ca2+ content [12].

4.3. Causes of the reduced effectiveness of Ca2+ induced Ca2+ release after CSQ over-expression
A concomitant reduction in RyR2 expression has previously been reported in some transgenic models [10]. But this is not the case in this study since maximal 3H Ry binding was similar in both groups (Fig. 1B). Alternatively, reduced sensitivity of CICR may be due to lowered lumenal free [Ca2+] after CSQ over-expression. Increased CSQ concentration and subsequent Ca2+ buffering may slow the rate of recovery of the free [Ca2+] within the lumen. If E–C coupling is initiated before a steady-state lumenal [Ca2+] is established, the lower [Ca2+] would reduce the open probability of the RyR [27–29]. However, this is not thought to be a likely mechanism in the present study for two reasons: (i) recent measurements of lumenal [Ca2+] signals in rabbit cardiomyocytes indicate that under similar conditions of stimulus frequency and temperature (0.5 Hz, 20 °C), steady state [Ca2+] within the SR is achieved rapidly during diastole [30]; (ii) the current study performed a limited set of experiments at stimulus frequencies as low as 0.25 Hz (data not shown), allowing a longer time between Ca2+ transients, CSQ over-expression did not result in larger SR Ca2+ release. However, this is only indirect evidence, therefore this mechanism cannot be completely discounted without concomitant measurements of luminal [Ca2+]. An alternative theory is that CSQ over-expression radically alters the geometry of dyadic junction and therefore the efficiency of coupling between the ICa,L and the SR Ca2+ release channel. No gross changes in cell shape and size were observed on CSQ over-expression (results not shown). However, the study of dyadic junctions would require the resolution of an electron microscope.

The proposal that CSQ over-expression has an inhibitory effect on the RyR2 is supported by studies on isolated RyR channels in bilayers. Addition of CSQ to the RyR/triadin/junctin complex in the presence of sub-millimolar [Ca2+] causes a reduction in the open probability of the channel [7,8]. The data would suggest that under control conditions, CSQ binding to the junction/triadin/RyR complex is not saturated, i.e. a significant amount of CSQ-free RyR/triadin/junction complexes must exist. Thus CSQ over-expression increases the number of CSQ-bound complexes and results in a decrease in overall RyR activity (decreased ‘gain’). A quite different effect would be anticipated if endogenous CSQ expression was higher, such that there were negligible amounts of CSQ-free complexes. Under these circumstances, CSQ over-expression would not be able to modulate RyR activity and the enhancement of Ca2+ buffering would be the sole effect. This may explain why a decreased E–C coupling gain was not evident in an earlier study on rat cardiomyocytes [12].

4.4. Effects of CSQ on Ca2+ sparks and SR Ca2+ content in permeabilised cardiomyocytes
In the present study, Ca2+ spark amplitude duration and frequency were unchanged yet SR Ca2+ content was increased after CSQ over-expression. Similarly, average Ca2+ spark amplitude was unchanged in the transgenic mouse model [11]. However in a recent study, adenoviral over-expression of CSQ in rat cardiomyocytes, increased amplitude of Ca 2+ sparks (frequency unchanged) [12]. The reason for this discrepancy is unknown and warrants further study. Increased SR Ca 2+ content is normally associated with increased Ca 2+ spark size and frequency [29]. The absence of significant changes suggests an inhibitory effect of CSQ over-expression on RyR function. This is consistent with the reduced E–C coupling gain observed in the intact cell.


   Acknowledgements
 
Expert technical assistance from Sandra Ott-Gebauer, Michael Kothe, Jessica Spitalieri, Aileen Rankin and Anne Ward is kindly acknowledged. This study was financed by grants from the British Heart Foundation and the German National Genome Research Network.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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