Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes
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 β-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. β-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.
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 (Po) 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
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 MΩ. [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).
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 (INCX) 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
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 INCX. 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).
Confocal Fluorescence Analysis and Calibration
Isolated rabbit cardiomyocytes were superfused with a mock intracellular solution and permeabilized with the use of β-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 ×60, 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
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.
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).
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 β-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/dtmax) 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 (ICa) 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 ICa amplitude, the time integral of ICa was calculated and converted to a Ca2+ influx (normalized to cell capacitance). Neither the amplitude of ICa 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 ICa. 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 ICa in these experiments.
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 INCX 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, INCX 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 INCX 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 INCX 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 INCX 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.
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 β-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 (Kd 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]).
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 INCX decay in response to caffeine, and (3) increased Ni2+-sensitive outward currents at membrane voltages positive to the reversal potential for INCX. 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 ICa
The LTCC current amplitude, time course, and I-V relationship were unaltered in sorcin-overexpressing cardiomyocytes. Recently, binding of sorcin to the α1-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 ICa 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 ICa 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 INCX (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 INCX 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+]i29,30; 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+-ATPase1,4). 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 INCX 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.
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.
Original received December 30, 2002; resubmission received April 30, 2003; revised resubmission received June 3, 2003; accepted June 3, 2003.
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