This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 93/2/132    most recent 01.RES.0000081596.90205.E2v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seidler, T. Articles by Smith, G. L. Search for Related Content PubMed PubMed Citation Articles by Seidler, T. Articles by Smith, G. L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CAFFEINE CALCIUM COMPOUNDS CALCIUM, ELEMENTAL NICKEL, ELEMENTAL Related Collections Contractile function Calcium cycling/excitation-contraction coupling Heart failure - basic studies Ion channels/membrane transport

(Circulation Research. 2003;93:132.)
© 2003 American Heart Association, Inc.

Cellular Biology

Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes

Tim Seidler, Stewart L.W. Miller, Christopher M. Loughrey, Astrid Kania, Annika Burow, Sarah Kettlewell, Nils Teucher, Stefan Wagner, Harald Kögler, Marian B. Meyers, Gerd Hasenfuss, Godfrey L. Smith

From the Department of Cardiology and Pneumology (T.S., A.K., A.B., N.T., S.W., H.K., G.H.), Georg-August-University Goettingen, Goettingen, Germany; Institute of Biomedical and Life Sciences (S.L.W.M., S.K., G.L.S.), University of Glasgow, Glasgow, UK; Institute of Comparative Medicine (C.M.L.), University of Glasgow Veterinary School, University of Glasgow, Glasgow, UK; and Department of Medicine (M.B.M.), Cardiology Division, New York University School of Medicine, New York, NY.

Correspondence to G. Hasenfuss, MD, Georg-August-Universität Goettingen, Abt. Kardiologie & Pneumologie, Robert-Koch-Strasse 40, 37075 Goettingen, Germany. E-mail hasenfus{at}med.uni-goettingen.de

	Abstract

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To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A ß-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37°C) was significantly reduced in Ad-sorcin–transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001). Action potential duration (at 20°C) was significantly less in the Ad-sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). In voltage-clamped, fura 2–loaded myocytes (20°C), a reduced peak-systolic and end-diastolic [Ca²⁺]_i was observed after Ad-sorcin transfection. L-type Ca²⁺ current amplitude and time course were unaffected. Caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na⁺-Ca²⁺ exchanger (NCX) current revealed a significantly lower SR Ca²⁺ content and faster Ca²⁺-extrusion kinetics in Ad-sorcin–transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. ß-Escin–permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca²⁺ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca²⁺] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca²⁺ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca²⁺-transient amplitude predominately by lowering SR Ca²⁺ content via increased NCX activity. The effect of sorcin overexpression on Ca²⁺ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.

Key Words: excitation-contraction coupling • calcium • cardiac myocytes • adenoviral gene transfer

	Introduction

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Tight and phase-dependent control of Ca²⁺ influx, efflux, and storage is a prerequisite for activation and relaxation of contractile activity during systole and diastole.¹ At the molecular level, Ca²⁺ influx across the sarcolemma is mediated by voltage-dependent activation of L-type Ca²⁺ channels (LTCCs). Ca²⁺ release from the sarcoplasmic reticulum (SR) subsequently occurs via clusters of SR Ca²⁺-release channels (ryanodine receptors [RyRs]). Opening of an individual RyR cluster causes a local transient increase of cytoplasmic [Ca²⁺], termed a Ca²⁺ spark.² Normal excitation-contraction (E-C) coupling involves Ca²⁺ influx via LTCCs, which initiate temporal and spatial synchronization of Ca²⁺ sparks, generating a global Ca²⁺ transient.³ The rise in [Ca²⁺]_i is terminated by both closure of RyRs and LTCCs and Ca²⁺ removal from the cytosol. The latter occurs through Ca²⁺ uptake into the SR by SR Ca²⁺-ATPase (SERCA) which competes with Ca²⁺ extrusion to the extracellular space via the Na⁺-Ca²⁺ exchanger (NCX), with a minor contribution from sarcolemmal Ca²⁺-ATPase.^1,4

The cardiac RyR isoform (RyR2) forms a macromolecular complex with numerous regulatory proteins,⁵ the functional effects of which are currently under investigation. One such protein is sorcin, a 22-kDa protein of the penta-EF hand Ca²⁺-binding protein family.⁶ Recent work has shown that sorcin associates with both the LTCC⁷ and RyR2.⁸ In lipid bilayers, sorcin was found to decrease the open probability (P_o) of RyR2,⁹ and on the basis of these findings, sorcin has been suggested to play a mechanistic role in the termination of RyR activation.^9,10 Information on the effect of sorcin on Ca²⁺ handling in adult cardiomyocytes is not available.

	Materials and Methods

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Adenoviral Overexpression of Sorcin in Isolated Adult Cardiomyocytes
The human sorcin gene in vector pCI-neo was cloned and ligated downstream from an immediate-early cytomegalovirus (CMV) promoter into vector pACCMV.pLpA using primers creating KpnI and HindIII sites. Recombination with pJM17 plasmid and production of replication-deficient adenovirus were carried out according to standard procedures.¹¹ Isolation and culture of adult rabbit ventricular cardiomyocytes were carried out as described.^12,13

Measurements of Protein Expression Levels and Cardiomyocyte Contractility
For Western blot protocols, see online data supplement (available at www.circres.ahajournals.org). Contractility data were recorded via video edge detection as described,¹² with cardiomyocytes superfused with pH-equilibrated Krebs-Henseleit solution containing 1.75 mmol/L Ca²⁺ and stimulated via parallel field electrodes at 1 Hz and 37°C. The shortening measurements were with the experimenter blinded to the group assignment.

Electrophysiological and [Ca²⁺]_i Measurements in Rabbit Cardiomyocytes
After 24-hour incubation with the virus, cardiomyocytes were superfused with a HEPES-based Krebs-Henseleit solution at 20°C to 21°C in a chamber on an inverted microscope. Current clamp or voltage clamp was achieved using the whole-cell ruptured-patch technique with an Axoclamp 2A amplifier (Axon Instruments). Pipette resistance was 7 to 10 M. [Ca²⁺]_i was measured from fura 2 fluorescence signals by use of a dual-wavelength spectrophotometric method described previously.¹⁴ Cytosolic loading of fura 2 was achieved by incubating cardiomyocytes with 5 µmol/L fura 2-AM at 20°C for 12 minutes (see online data supplement).

Electrophysiology Protocols
Current-Clamp Measurements
Action potentials (APs) were elicited in cardiomyocytes by current injection (1 nA, 5 ms) every 1 second. The microelectrodes were filled with (mmol/L) KCl 120, NaCl 10, HEPES 10, and EGTA 0.1 (pH 7.0). Once in the steady state, the average of 8 sequential AP recordings was acquired and analyzed.

E-C Coupling Protocol
Rabbit cardiomyocytes were held at -80 mV, and the voltage was stepped to -40 mV (50 ms) to inactivate the inward Na⁺ current, before stepping to 0 mV (150 ms) and returning to -80 mV. This protocol was repeated every second for 1 minute to achieve steady-state Ca²⁺ transients. SR Ca²⁺ content and NCX activity were then estimated by rapidly switching to 10 mmol/L caffeine to cause SR Ca²⁺ release. In the continued presence of caffeine, the SR is unable to reaccumulate Ca²⁺, and elimination of Ca²⁺ is mainly due to NCX (Figure 3). The time course of the decay of [Ca²⁺] and the NCX-mediated inward current (I_NCX) represent rates of extrusion of Ca²⁺ from the cell predominately by NCX.¹⁵ These signals were fitted to exponential decays over >80% of their amplitude. The magnitude of non-NCX Ca²⁺-removal mechanisms was estimated from the Ca²⁺ decay obtained by rapidly switching to 10 mmol/L caffeine in the presence of 10 mmol/L NiCl₂.¹⁵

View larger version (33K): [in this window] [in a new window]   Figure 3. Characteristics of caffeine-induced SR Ca2+ release and the corresponding membrane currents recorded on rapid application of caffeine. A, Registrations of [Ca2+]i and membrane current recorded on application of 10 mmol/L caffeine (as indicated above the records) in LacZ-transfected (i) and sorcin-transfected (ii) cardiomyocytes. Bi, Superimposed decline phase of [Ca2+]i record from sorcin-transfected (open circle) and LacZ-transfected cells (extract from panel A). Bii, Superimposed records of the decline phase of membrane currents in response to rapid application of caffeine in sorcin-transfected (open circle) and LacZ-transfected cells (extract from panel A). C, Mean±SEM values: peak [Ca2+]i for LacZ (n=21) and sorcin (n=17) groups, P<0.01 (i); peak INCX for LacZ (n=18) and sorcin (n=13) groups, P=NS (ii); and INCX · time integral, LacZ (n=18), and sorcin (n=13) groups, P<0.01 (iii). D, Mean±SEM values: rate constant for the decay of the caffeine-induced Ca2+ transient for LacZ (n=17) and sorcin (n=12) groups, P<0.01 (i); rate constant for the decay of the caffeine-induced Ca2+ transient in the presence of 10 mmol/L Ni2+ for LacZ (n=5) and sorcin (n=4) groups, P=NS (ii); and rate constant for the recovery of membrane current in response to caffeine for LacZ (n=11) and sorcin (n=8) groups, P<0.01 (iii).

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.¹⁶ Complete inhibition of the SR was achieved after 100-second perfusion; rapidly switching to 10 mmol/L caffeine did not generate Ca²⁺ release or I_NCX. Shorter periods of perfusion of thapsigargin achieved intermediate caffeine responses representing intermediate SR Ca²⁺ contents. Separate groups of cells were exposed to 20-, 40-, and 100-second periods of perfusion with thapsigargin; Ca²⁺-transient amplitude was measured from the last four transients before caffeine application.

NCX Current Density
After achieving the whole-cell configuration, a period of 4 to 5 minutes was allowed for dialysis of the pipette solution into the cell. Currents were then measured in response to a 3-second ramp from -120 to 80 mV from a holding potential of -80 mV. An ascending ramp was chosen because this has been shown to cause less perturbation of subsarcolemmal [Ca²⁺] than a descending ramp, and the resulting currents are closer to those obtained when a voltage step protocol is used.¹⁷ The ramp protocol was performed at 0.1 Hz until steady-state currents were achieved, whereupon data from five ramps were 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 (Ni²⁺-sensitive current) (Figure 4A).

View larger version (16K): [in this window] [in a new window]   Figure 4. Measurement of I-V relationship of Ni2+-sensitive (NCX) current. A, Ramp protocol used to generate I-V relationship. Voltage ramps from -120 to 80 mV were used under control conditions and in the presence of 5 mmol/L Ni2+ (i). The difference current (Im) (ii) represents the Ni2+-sensitive current nominally attributed to INCX. B, Average I-V relationship measured in LacZ and sorcin cells. C, Magnitude of currents measured at -120 and 80 mV in LacZ and sorcin groups. *P<0.01.

Confocal Fluorescence Analysis and Calibration
Isolated rabbit cardiomyocytes were superfused with a mock intracellular solution and permeabilized with the use of ß-escin (Sigma) (see online data supplement). Confocal line-scan images were recorded by use of a Bio-Rad Radiance 2000 confocal system. Fluo 3 or fluo 5F (10 µmol/L) in the perfusing solution was excited at 488 nm (Kr laser) and measured >515 nm with the use of epifluorescence optics of an inverted microscope with a water-immersion objective lens (magnification x60, numerical aperture 1.2). To enable this trace to be converted to [Ca²⁺], a series of calibration solutions was used at the end of each Ca²⁺-spark measurement period incorporating 10 mmol/L EGTA, as previously described¹³ (see online data supplement). In all experiments, the [Ca²⁺] in the test solution was 145 to 160 nmol/L. Ca²⁺ sparks recorded in fluo 3–containing solutions were quantified by use of an automatic detection and measurement algorithm adapted from a previously published method.¹⁸

Statistical Analysis
Data were expressed as mean±SEM. For contractility, ion currents, intracellular [Ca²⁺], and Ca²⁺-spark parameters, comparisons were performed using the unpaired Student t test; otherwise, a paired Student t test was used, and differences were considered significant at P<0.05. ANOVA with a Tukey post hoc test was used for multiple comparisons.

	Results

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mRNA, Protein, and Contractility Measurements in Adult Rabbit Cardiomyocytes
After infection of cardiomyocytes with adenovirus encoding for sorcin, mRNA and protein levels rose in a dose-dependent manner, as demonstrated by reverse transcriptase (RT)–polymerase chain reaction (PCR) (Figure 1Ai) and Western blots (Figure 1Aii). Calsequestrin levels were used as a standard in both RT-PCR and Western blots. Separate measurements confirmed that protein levels of NCX and calsequestrin remained equal to those of untransfected myocytes 48 hours after infection with up to 100 multiplicity of infection (MOI) Ad-sorcin (not shown).

View larger version (35K): [in this window] [in a new window]   Figure 1. A, Verification of transgene expression in isolated adult rabbit cardiomyocytes. Forty-eight hours after transfection with indicated MOIs (numbers), RT-PCR (i) and Western blots (ii) indicate overexpression of sorcin mRNA and protein. -RT indicates control reaction without RT. Detection of calsequestrin (CSQ) served to demonstrate equal loading. B, Fractional shortening (FS) of isolated adult cardiomyocytes. At 48 hours after transfection with Ad-sorcin at MOI 100, FS is reduced in rabbit cardiomyocytes compared with control Ad-LacZ–transfected cardiomyocytes (Ad-LacZ, n=311; Ad-sorcin, n=311; *P<0.001). C, AP recordings (20°C to 21°C) from isolated cardiomyocytes from Ad-LacZ and Ad-sorcin groups (i). AP duration was decreased after infection with Ad-sorcin. Mean APD90 is shown (ii).

In isolated adult rabbit cardiomyocytes stimulated at 1 Hz 48 hours after infection with Ad-Sorcin, fractional shortening was reduced by 15% when those myocytes were compared with ß-galactosidase (Ad-LacZ)–transfected cardiomyocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001) (Figure 1B).

Measurement of AP Characteristics in Rabbit Myocytes
Figure 1Ci shows superimposed APs recorded at 20°C to 21°C from single cardiomyocytes after Ad-LacZ or Ad-sorcin transfection. As indicated, the AP duration was shorter in myocytes after Ad-sorcin transfection. On average, the AP duration at 90% repolarization (APD₉₀) was significantly less in the Ad-sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). Other parameters (resting membrane potential, dV/dt_max) were not significantly altered.

[Ca²⁺]_i Measurements in Voltage-Clamped Rabbit Cardiomyocytes
The reduced cardiomyocyte contractility was paralleled by a significant decrease in peak-systolic [Ca²⁺] and end-diastolic [Ca²⁺] in voltage-clamped rabbit cardiomyocytes after Ad-sorcin transfection (Figures 2A and 2C), resulting in a smaller Ca²⁺-transient amplitude. As illustrated in Figure 2A, Ca²⁺ current (I_Ca) amplitude was monitored by incorporating a prepulse to -40 mV for 50 ms to inactivate the inward Na⁺ current. In addition to the measurements of I_Ca amplitude, the time integral of I_Ca was calculated and converted to a Ca²⁺ influx (normalized to cell capacitance). Neither the amplitude of I_Ca nor the integral of the current was different between the two experimental groups (Figures 2B and 2D). These measurements were confirmed by separate experiments examining the current-voltage (I-V) relationship of I_Ca. In these experiments, [Ca²⁺]_i was lowered and buffered by dialysis of the cardiomyocyte with 5 mmol/L BAPTA. As shown in the online data (online Figure 1, available at http://www.circresaha.org), no difference in the I-V relationship was observed, confirming the absence of an effect of sorcin overexpression on the I_Ca in these experiments.

View larger version (23K): [in this window] [in a new window]   Figure 2. Depolarization-induced Ca2+ transients recorded from Ad-LacZ– and Ad-sorcin–transfected cardiomyocytes. A, Records of membrane voltage (Em), membrane current, and [Ca2+]i from single cardiomyocytes (average of 10 signals). B, Membrane current recorded on depolarization from the prepulse potential of -40 to 0 mV for 150 ms to illustrate amplitude and time course of the LTCC (extracts from panel Ai and Aii). C, Mean±SEM values of peak-systolic [Ca2+]i (i) and end-diastolic [Ca2+]i (ii). D, Mean±SEM values of LTCC amplitude (i) and Ca2+ influx via LTCC (current-time integral/96 500C · mol-1) (ii).

SR Ca²⁺ Content as Assessed by 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⁺] 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 were monitored together with the Ca²⁺ transient (Figures 3A and 3Ci). As shown in Figure 3Ci, the peak of the caffeine-induced Ca²⁺ release was significantly smaller in Ad-sorcin–transfected cardiomyocytes, suggesting a reduced SR Ca²⁺ content (Ad-LacZ, 895±70 nmol/L, n=21; Ad-sorcin, 528±32 nmol/L, n=17; P<0.01). However, the peak of the transient inward current was not significantly smaller in Ad-sorcin–transfected cells (Figure 3Cii). If it is assumed that the caffeine-induced transient inward current is entirely due to the activity of NCX, the time integral of current 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). As shown in Figure 3Ciii, the mean integral of I_NCX in the sorcin-transfected group was significantly lower than that in the control (LacZ-transfected) group (normalized to cell capacitance), supporting the conclusion that SR Ca²⁺ content was significantly reduced in sorcin-overexpressing cardiomyocytes.

Sarcolemmal Ca²⁺-Efflux Rates in Rabbit Cardiomyocytes
As shown in Figure 3Cii, I_NCX amplitude was similar in Ad-sorcin and Ad-LacZ groups; the smaller time integral in the sorcin group (Figure 3Ciii) indicates a faster decay of the inward current. Examples of the time course of the inward current decay and the corresponding decrease in [Ca²⁺] are shown in Figures 3Bi and 3Bii; both I_NCX and [Ca²⁺] decayed faster in Ad-sorcin–transfected cardiomyocytes. These decays were fitted to a single exponential, and mean rate constants were calculated (Figure 3D). The significantly higher rate constants for [Ca²⁺] decay (Figure 3Di) and I_NCX decay (Figure 3Diii) indicate a more rapid extrusion of Ca²⁺ via NCX in Ad-sorcin–transfected cells. In separate experiments, the rate of decay of [Ca²⁺]_i was measured in response to caffeine application in the presence of 10 mmol/L Ni²⁺. Under these conditions, NCX is inhibited, and the decay of [Ca²⁺] represents Ca²⁺ extrusion via sarcolemmal Ca²⁺-ATPase and possibly mitochondrial uptake.^19,20 Importantly, under these conditions, the rate constant for decay was not significantly different between the two experimental groups, indicating that Ca²⁺ extrusion via non–NCX-mediated mechanisms was unaffected by sorcin overexpression (Figure 3Dii).

Relationship Between SR Ca²⁺ Content and Ca²⁺-Transient Amplitude in Rabbit Cardiomyocytes
To determine whether the decreased SR Ca²⁺ content can account quantitatively for the decreased Ca²⁺-transient amplitude, measurements were made with the use of thapsigargin to progressively decrease SR Ca²⁺ content. As shown in Figure 5, the plot of I_NCX integral and Ca²⁺-transient amplitude for the Ad-LacZ group generated an approximately hyperbolic relationship. These data from the Ad-sorcin group can be superimposed on the relationship described for the LacZ group. These data suggest that within the limits of the measurements, the effects of sorcin overexpression on E-C coupling could be explained by a decrease in SR Ca²⁺ content alone, with no evidence for additional effects on E-C coupling gain.

View larger version (30K): [in this window] [in a new window]   Figure 5. Relationship between INCX integral (an index of SR Ca2+ content) and Ca2+-transient amplitude for cardiomyocytes from Ad-LacZ (filled symbols) and Ad-sorcin (open symbols) groups. Control data (circles) were obtained in the steady state; lower SR loads were achieved by exposure to 5 µmol/L thapsigargin for various times as indicated in the inset. The current integral (in coulombs, C) was expressed relative to the cell capacitance (in farads, F) to normalize for variations in cell volume.

NCX I-V Relationship
NCX activity was investigated further by measuring the Ni²⁺-sensitive current generated by slow ascending ramp voltages from -120 to 80 mV (Figures 4Ai and 4Aii). As shown previously, this protocol can be used to measure the I-V relationship on NCX.¹⁷ [Ca²⁺]_i was buffered at 250 nmol/L using 50 mmol/L EGTA in the patch-pipette solution. Higher than normal [Na⁺]_i (20 mmol/L) was used to standardize the [Na⁺]_i. The average Ni²⁺-sensitive currents generated by this protocol were recorded (Figure 4B). In these experiments, larger currents were measured in Ad-sorcin–transfected cardiomyocytes than in control (LacZ-transfected) myocytes; the difference reached significance at positive potentials (10 to 80 mV). There was a trend toward larger currents at -120 mV in Ad-sorcin–transfected cardiomyocytes, but the difference did not achieve significance (P=0.08, Figure 4C).

Ca²⁺-Spark Activity and Caffeine Responses in Permeabilized Rabbit Cardiomyocytes
Investigating possible direct effects of sorcin overexpression on the SR of intact cardiomyocytes is complicated by a sorcin-mediated increase in NCX activity and the rapid loss of Ca²⁺ from the SR during the quiescent periods required for Ca²⁺-spark recording. For this reason, sarcolemmal fluxes were functionally bypassed by the permeabilization of the sarcolemma with ß-escin. Under these circumstances, single cardiomyocytes can be superfused with a standardized [Ca²⁺] (150 nmol/L) and pH (7.0) in the presence of ATP and creatine phosphate to maintain SR function. The permeabilization allows complete equilibration of the cytosol with the [Ca²⁺] of the superfusion medium. Inclusion of fluo 3 (10 µmol/L) allows Ca²⁺-spark activity to be monitored by laser-scanning confocal microscopy. SR Ca²⁺ content was assessed by rapid application of 10 mmol/L caffeine. Figures 6Ai and 6Aii show line-scan images recorded from permeabilized rabbit cardiomyocytes after transfection with Ad-LacZ or Ad-sorcin, respectively. Ca²⁺ sparks were evident as transient increases of relative fluorescence (F/F₀); a 10-pixel band from each line scan (marked by arrows in Figure 6A) is shown in Figure 6B for clarity. The Ca²⁺ sparks recorded in Ad-sorcin–transfected cardiomyocytes were smaller than those recorded in the Ad-LacZ control myocytes, and the average Ca²⁺-spark peak, frequency, width, and duration (Figures 6Ci to 6Civ) were all significantly reduced in the sorcin-overexpressing cells (peak F/F₀ 1.96±0.02 versus 1.84±0.04, P<0.05; frequency 0.058±0.004 versus 0.040±0.004 µm^-1s^-1, P<0.05; width 3.30±0.06 versus 3.06±0.06 µm, P<0.05; duration 28.2±1.0 versus 24.8±1.2 ms, P<0.05; and n=21 cells for Ad-LacZ versus n=11 cells for Ad-sorcin). Despite the reduced frequency of measured Ca²⁺ sparks, the distribution histograms for amplitude, width, and duration were of similar shape between the two experimental groups (see online Figure 2, available at http://www.circresaha.org). This suggests that sorcin overexpression did not result in a development of a distinct subtype of Ca²⁺-spark event, which would be evident as a distortion of the distribution histograms. After perfusion with nominally 150 nmol/L Ca²⁺ (50 µmol/L EGTA), 10 mmol/L caffeine was rapidly applied, and the cardiomyocyte was subsequently exposed to a series of calibration solutions (see online data supplement). This procedure ensured that the [Ca²⁺] within the solutions used to monitor Ca²⁺-spark activity contained 145 to 160 nmol/L Ca²⁺. These experiments were repeated using the low-affinity dye fluo 5F (K_d 1.03 µmol/L) to quantify the SR Ca²⁺ content by measuring the mean amplitude and time integral of the Ca²⁺ signal on rapid application of caffeine (Figure 6Di). No significant difference in SR content was measured in permeabilized cardiomyocytes overexpressing sorcin (Figure 6Dii; peak [Ca²⁺] in caffeine was 1.17±0.14 µmol/L for Ad-LacZ [n=12] and 1.15±0.13 µmol/L for Ad-sorcin [n=13]).

View larger version (43K): [in this window] [in a new window]   Figure 6. Ca2+ sparks in permeabilized cardiomyocytes transfected with LacZ (control) and sorcin. A, Pseudo color line scan epifluorescence image from single permeabilized cardiomyocytes after transfection with LacZ and sorcin. B, Plots of F/F0 (average of a band of 5 pixels) taken from the images in panel A at the points shown by the arrows. C, Mean±SEM values for peak F/F0 (i), spark frequency (ii), spark width (iii), and spark duration (iv). Results were compiled from 1844 events from 21 cells (Ad-LacZ) and 739 events from 11 cells (Ad-sorcin). *P<0.01. D, [Ca2+] signal calculated from the mean fluo 5F fluorescence signal of a 20-pixel intracellular region on application of 10 mmol/L caffeine with the accompanying signal integral (i) and mean values of peak [Ca2+] and integral signals in response to caffeine in the 2 experimental groups (ii).

	Discussion

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Decreased contractility was noted in field-stimulated adult rabbit cardiomyocytes overexpressing sorcin. The present study examines the cellular mechanisms responsible for this effect of sorcin on cardiac E-C coupling.

Effects of Sorcin on NCX Activity
A novel observation reported in the present study is that NCX activity is increased in sorcin-overexpressing cardiomyocytes. This conclusion is based on the following: (1) increased rate constant of [Ca²⁺] decay in response to caffeine in intact cardiomyocytes, (2) increased rate constant of I_NCX decay in response to caffeine, and (3) increased Ni²⁺-sensitive outward currents at membrane voltages positive to the reversal potential for I_NCX. This hypothesis is further supported by the significant reduction in end-diastolic [Ca²⁺] consistent with enhanced extrusion of Ca²⁺ during diastole (at -80 mV) via NCX. Notably, Ca²⁺ extrusion via an Ni²⁺-insensitive mechanism was unaffected by sorcin overexpression. The cellular mechanism underlying sorcin-induced increase in NCX activity is unknown.

Effects of Sorcin on I_Ca
The LTCC current amplitude, time course, and I-V relationship were unaltered in sorcin-overexpressing cardiomyocytes. Recently, binding of sorcin to the ₁-subunit of LTCC has been demonstrated,⁷ and a preliminary report has suggested that sorcin may modulate LTCCs in cardiomyocytes.²¹ Although the data presented in the present study do not exclude a role of sorcin in modulating LTCC under other conditions, altered LTCC function does not seem to contribute to the observed reduction of intracellular Ca²⁺ reported in the present study. The cellular distribution of sorcin between membranes and cytosol has been reported to depend on [Ca²⁺]_i.²² Therefore, the effects of sorcin on I_Ca may depend on the concentration of sorcin and Ca²⁺ in the vicinity of the LTCC. The peak systolic [Ca²⁺] in cultured cardiomyocytes is considerably lower than that in freshly dissociated cells; thus, Ca²⁺-dependent inactivation of the current may be minimal in these cells. Under these conditions, the ability of sorcin to affect I_Ca may be substantially reduced.

Effect of Sorcin on AP Duration
Decreased AP duration was observed in the Ad-Sorcin group compared with the control group. This effect would contribute to a negative inotropic effect by increasing the period of time at negative membrane potentials between stimuli, thereby promoting Ca²⁺ efflux via NCX.²³ However, preliminary voltage-clamp studies suggest that reduction of APD₉₀ alone (by 20%) would not contribute significantly to the reduced Ca²⁺-transient amplitude observed in the Ad-sorcin group (data not shown). Alternatively, changes in the early phase of repolarization are known to influence E-C coupling via changes in LTCC amplitude.²⁴ This latter mechanism may contribute to the negative inotropy observed in the present study. The ionic basis for the reduced AP duration is not known; previous studies using adenovirus-mediated NCX overexpression in the rabbit noted no major changes in AP duration²⁵; therefore, further work is required in investigating the basis of the electrophysiological effects.

E-C Coupling Studies in Voltage-Clamped Rabbit Cardiomyocytes
The reduced amplitude of the Ca²⁺ transient in voltage-clamped myocytes and reduced Ca²⁺ release in response to caffeine suggest that reduced SR Ca²⁺ content is a significant contributor to the negative inotropic effect independent of changes in AP shape. However, it is not clear whether the decrease of SR content in sorcin-overexpressing cells can account quantitatively for the decrease of the Ca²⁺ transient. This was established by investigating whether the relationship between SR Ca²⁺ content and Ca²⁺ amplitude is the same for both Ad-LacZ and Ad-sorcin cells. The approximately hyperbolic relationship between the integral of I_NCX (an index of SR Ca²⁺ content) and Ca²⁺-transient amplitude in the Ad-LacZ group indicated by the data in Figure 5 has been observed by others in both rat and rabbit cardiomyocytes.^26,27 Notably, the lower I_NCX integral and smaller Ca²⁺-transient amplitude observed in the Ad-sorcin group were very similar to values achieved in the Ad-LacZ group after reduction of the SR Ca²⁺ content using thapsigargin (Figure 5). Similarly, reducing SR Ca²⁺ content in myocytes from the Ad-sorcin group generated a data point that lay on the relationship described by the LacZ group. This strongly suggests that the predominant cause of the reduced Ca²⁺ transient in the Ad-sorcin group is reduced SR Ca²⁺ content. If sorcin overexpression had reduced sensitivity of the RyR for Ca²⁺, the relationship between SR Ca²⁺ content and Ca²⁺-transient amplitude would have been shifted to the right of control group.²⁸

Effects of Sorcin on Ca²⁺-Spark Characteristics in Permeabilized Myocytes
Previous studies have shown a direct effect of sorcin on isolated RyR activity. An indirect measure of Ca²⁺ efflux via RyR can be assessed by measuring the maximum rate of change of [Ca²⁺]_i during a Ca²⁺ transient. This parameter was not different in the Ad-LacZ group compared with the Ad-sorcin group (2.93±0.27 µmol/L per second [n=41] versus 2.87±0.27 µmol/L per second [n=44], respectively). A more direct measure of RyR activity is the recording of Ca²⁺ sparks with the use of confocal microscopy. Measurement of Ca²⁺-spark activity in intact rabbit cardiomyocytes in the steady state is problematic because [Ca²⁺]_i and SR Ca²⁺ content rapidly decrease on cessation of stimulation due to net sarcolemmal efflux. Ca²⁺-spark parameters are very sensitive to [Ca²⁺]_i^29,30; therefore, interpretation of results is difficult without knowledge and/or control of the [Ca²⁺]_i in cells from different experimental groups. The marked increase in NCX activity observed in the Ad-sorcin group would reduce SR Ca²⁺ content and diastolic [Ca²⁺], thereby altering Ca²⁺-spark activity indirectly. For these reasons, Ca²⁺-spark activity was monitored in permeabilized cardiomyocytes at a precisely measured bathing [Ca²⁺]. Previous work has established that Ca²⁺-spark characteristics in permeabilized cells are indistinguishable from those observed in intact cells and regulated by known modulators of RyR2 activity (Ca²⁺/calmodulin and cADP ribose) but with the added advantage that cytoplasmic conditions can be standardized.²⁹ The present data show that whereas SR Ca²⁺ loading is comparable in permeabilized myocytes from Ad-sorcin and Ad-LacZ groups, Ca²⁺-spark characteristics differ significantly. Ca²⁺-spark amplitude, width, duration, and frequency were all significantly reduced. This result is consistent with previous work indicating that (1) sorcin coimmunoprecipitates with RyR2,⁸ and (2) RyR2 closed time is prolonged and burst frequency is reduced in single-channel recordings of swine RyR2 in planar lipid bilayers on the addition of recombinant sorcin.⁹ As indicated in Figure 5, this effect was not associated with a detectable change in the E-C coupling efficiency in intact myocytes under the conditions used in the present study. However, this effect on RyR2 activity may have a significant impact on E-C coupling efficiency at different stimulus rates and temperatures.

Can Increased NCX Activity Account for Decreased Ca²⁺-Transient Amplitude?
In the steady state during repetitive stimulation, Ca²⁺ influx through LTCCs equals Ca²⁺ extrusion by NCX (with a minor component via the sarcolemmal Ca²⁺-ATPase^1,4). The balance between influx and efflux can be achieved in sorcin-overexpressing cells at a lower [Ca²⁺]_i, because less cytosolic Ca²⁺ is required to generate equivalent Ca²⁺ efflux on NCX compared with control cardiomyocytes. In support of this analysis, specific upregulation of NCX has previously been linked to reduced SR Ca²⁺ content and negative inotropy.³¹ In the present study, the rate constant for Ca²⁺ efflux is increased by 50% on sorcin overexpression. By use of a simple model of Ca²⁺ fluxes, this increase would be expected to reduce diastolic [Ca²⁺] to 66% of the control value. This agrees well with the decrease to 68% of the control values reported (Figure 2Cii). Again, by use of simplifying assumptions concerning SR Ca²⁺ flux and buffering, these changes would be expected to lower SR Ca²⁺ content to 66% of the control values. This agrees well with the decrease in I_NCX integral to 70% of the control values (Figure 3Diii). Thus, the increased NCX activity alone appears to be sufficient to account for the reduced SR Ca²⁺ content observed.

Effect of Sorcin Overexpression on Cardiac Inotropy
The data in the present study suggest that reduced SR Ca²⁺ content causes the negative inotropic effects of sorcin overexpression. However, direct comparison of the relative cell shortening and [Ca²⁺]_i cannot be made easily because the measurements were made separately and under different experimental conditions. Increased sorcin levels reduced cellular Ca²⁺ load that was predominately due to an increased NCX activity. The consequence of this effect may be an improved diastolic function, leading to preservation of intracellular high-energy phosphate levels. Direct effects of sorcin overexpression on RyR2 activity were noted, but the consequence of this on E-C coupling was not evident under these conditions. In addition to direct effects on E-C coupling, the long-term effects of sorcin-mediated alterations in [Ca²⁺]_i levels may include altered cardiac gene expression.

	Acknowledgments

 
This study was supported by grants from the German National Genome Research Network (NGFN), the Deutsche Forschungsgemeinschaft, and the British Heart Foundation. An Engineering and Physical Sciences Research Council studentship and the School of Veterinary Medicine, Glasgow University, support C.M. Loughrey; a British Heart Foundation studentship supports S.L.W. Miller. Dr Meyers is supported by a Kenner Grant-in-Aid from the American Heart Association. Expert technical assistance from Sandra Ott-Gebauer, Jessica Spitalieri, Michael Kothe, Aileen Rankin, and Anne Ward is kindly acknowledged.

	Footnotes

 
Original received December 30, 2002; resubmission received April 30, 2003; revised resubmission received June 3, 2003; accepted June 3, 2003.

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