Ca2+ Signaling in Cardiac Myocytes Overexpressing the α1 Subunit of L-Type Ca2+ Channel
Voltage-gated L-type Ca2+ channels (LCCs) provide Ca2+ ingress into cardiac myocytes and play a key role in intracellular Ca2+ homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca2+ signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the α1 subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell ICa density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca2+ release, the “gain” function of EC coupling was uncompromised, and SR Ca2+ content, diastolic cytosolic Ca2+, and unitary properties of Ca2+ sparks were unchanged. Importantly, the enhanced ICa entry and SR Ca2+ release were associated with an upregulation of the Na+-Ca2+ exchange activity (indexed by the half decay time of caffeine-elicited Ca2+ transient) by 27% and SR Ca2+ recycling by ≈35%. Western analysis detected a 53% increase in the Na+-Ca2+ exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of ICa kinetics suggested that SR Ca2+ release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca2+ signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca2+ influx triggers a coordinated remodeling of Ca2+ handling to maintain Ca2+ homeostasis.
As a ubiquitous intracellular messenger, Ca2+ plays a pivotal role in many biological processes, including muscle contraction, gene regulation, enzymatic reactions, cell survival, and cell death.1,2⇓ In the heart, voltage-operated L-type Ca2+ channels (LCCs) provide the critical pathway that gates the influx of Ca2+ into the cytoplasm from the exterior.2 Ca2+ entry triggers Ca2+ release from Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR), via the Ca2+-induced Ca2+ release mechanism.3 Proteins that also contribute to maintaining Ca2+ homeostasis and orchestrating the dynamic Ca2+ oscillations that drive contraction and relaxation of the heart, include the Na+-Ca2+ exchanger (NCX)4 and Ca2+-ATPase located in the sarcolemma (SL), SERCA2 and its regulator phospholamban (PLB)5,6⇓ in the SR, and calsequestrin, a low affinity, high capacitance Ca2+ buffer protein inside the SR lumen.1,4,7⇓⇓
The prevalent theory of cardiac excitation-contraction (EC) coupling is that SR Ca2+ release occurs in a discontinuous and discrete manner, ie, as Ca2+ sparks8 each involving the coordinated activation of 4 to 6 RyRs.9,10⇓ Ignition of Ca2+ sparks is under the tight control of local Ca2+ emitted from single LCCs.9–13⇓⇓⇓⇓ During relaxation, cytosolic Ca2+ is sequestered to the SR by SERCA2 and extruded out of the cell mainly by the SL NCX.10 It has been increasingly appreciated that, in addition to driving the heart beat, an increase in intracellular Ca2+ is involved in cardiac hypertrophy, apoptosis, and cardiac remodeling,14–16⇓⇓ although the specific underlying signaling mechanisms are not well understood. The density of the LCC in human and animal models of hypertrophy is still controversial with some reporting increases, others no change, and one a depression (see Muth et al2,14⇓ and references cited therein). What has been generally accepted for some years, however, is that changes in [Ca2+]i for the most part increase due to defects in the “handling” systems and are associated with the development of hypertrophy and eventual failure in human heart.17
The α1 subunit of LCC is the main structural and functional component, ie, the pore of the channel complex; the auxiliary subunits probably modulate gating and trafficking.2,18⇓ To fully explore the role of LCC-mediated Ca2+ influx in acute and chronic cardiac physiology and pathophysiology, we have generated a line of transgenic mice (Tg) with cardiac-specific overexpression of the human α1 subunit of the LCC.19 Tg mouse hearts are modestly hypertrophied at 4 months of age, and the β-adrenergic signaling pathway is blunted. Apoptosis is detectable in Tg hearts at 8 months of age. The mice develop full-blown cardiomyopathy and heart failure around the 10 to 12 month period and die of congestive heart failure. The gradually developing disease process resembles the human disease process of dilated cardiomyopathy.2,14⇓ Thus, this line of Tg mice provides a useful tool to study how a single primary perturbation of Ca2+ homeostasis leads to the profound end-effect of cardiomyopathy.
In the present study, we have systematically characterized the main components of Ca2+ regulation in Tg and nontransgenic (Ntg) hearts to gain insights into adaptation of Ca2+ handling in response to a sustained perturbation of Ca2+ influx. We opted to use the 4-month-old Tg animals, when the hearts are at threshold hypertrophy. Our results indicate that, in spite of the modest cardiac hypertrophy, both orthograde and retrograde Ca2+ signaling between LCCs and RyRs are preserved. More importantly, a sustained but modest increase in Ca2+ influx triggers a coordinated remodeling of Ca2+ handling to maintain Ca2+ homeostasis.
Materials and Methods
Recording of Whole-Cell ICa
Single ventricular myocytes were isolated from the hearts of 4-month-old Tg and Ntg littermates, using an enzymatic method described previously.20 The animal use protocols were approved by our institution’s Animal Care and Use Committee. Whole-cell patch-clamp technique was employed in conjunction with confocal linescan imaging. Patch pipette filling solution contained (in mmol/L) 120 CsCl, 10 NaCl, 10 tetraethylammonium chloride, 5 MgATP, and 20 HEPES (pH 7.2 adjusted with CsOH), plus 160 μmol/L fluo-3 pentapotassium salt (Molecular Probes). Bath solution for ICa recording contained (in mmol/L) 137 NaCl, 10 CsCl, 1.2 MgCl2, 15 Glucose, 10 HEPES, 2 CaCl2, and 0.02 TTX (pH 7.4). The ICa was activated by a series of 200-ms depolarization pulses from −50 mV holding potential to test potentials ranging from −40 mV to +60 mV at 10-second intervals. Four conditioning pulses of 100 ms and 0 mV were delivered at 1.0 Hz prior to each test pulse.
Single-channel recordings were performed using a cell-attached patch-clamp method described previously.21 Cells were equilibrated in a high potassium depolarizing solution: (in mmol/L) 120 K-Aspartate, 25 KCl, 10 HEPES, 10 Glucose, 2 MgCl2, 1 CaCl2, 2 EGTA, and 6 KOH (pH 7.2). The patch pipettes (8 to 20 MΩ) were filled with the pipette solution: (in mmol/L) 5 BaCl2, 5 4-aminopyridine, 10 HEPES, 300 sucrose, and 5 TEA-OH (pH 7.4).
Unitary Ca2+ current data were filtered at 1 kHz and sampled at 10 kHz using Axon PClamp software. After correction for capacitative and leakage currents, data were then passed through a 1-kHz digital Gaussian filter and converted into idealized events lists based on a 50% of unitary amplitude threshold criterion. Amplitude histograms were fitted to Gaussian distributions using PStat software (Axon Instruments, Inc) with the Simplex least-squares procedure.
Confocal Ca2+ Imaging
Ca2+ transients and sparks were measured using Zeiss LSM-410 confocal microscopy. The Ca2+ level was reported as F/F0, where F0 is the resting or diastolic fluo-3 fluorescence, or calculated using the equation8 C=Kd×R/(Kd/C0+1−R), where R denotes F/F0, Kd (=1.1 μmol/L) the dissociation constant of fluo-3 in the cytoplasm, and C0 (=150 nmol/L) the resting Ca2+ level. The detection and parametric measurement of Ca2+ sparks in the line-scan images were automated with the aid of a computer algorithm coded in IDL.22 SR Ca2+ content was assessed by measuring caffeine-induced Ca2+ transient, in which 20 mmol/L caffeine was applied rapidly by a picospritzer. All experiments were performed at room temperature (22 to 24°C).
The homogenates from Tg and Ntg hearts were sonicated to fragment the nucleic acids, boiled for 10 minutes, centrifuged for 10 minutes at 13 500 rpm (4°C), and then the supernatants were removed. Cardiac homogenates (25 μg total protein for RyR, SERCA2, and PLB and 75 μg for NCX) were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes for RyR, SERCA2, and PLB blotting and to nitrocellulose for NCX blotting. Blots were probed with the following primary antibodies: mouse monoclonal anti-RyR, mouse monoclonal anti-PLB, mouse monoclonal anti-SERCA (all 3 from Affinity Bioreagents Inc), and mouse monoclonal anti-NCX (Swant). Anti-mouse peroxidase-conjugated secondary antibodies (Boehringer Mannheim) were added. Protein bands were quantitated using Scion Image software (National Institutes of Health).
Data are reported as mean±SEM. Student’s t test and ANOVA for repeated measures were applied, when appropriate, to determine statistical differences. A value of P<0.05 was considered statistically significant.
Enhanced SL Ca2+ Influx in Tg Myocytes
Figure 1A shows 2 families of ICa traces elicited in a representative Ntg and Tg myocyte, respectively. The average peak ICa at 0 mV was −1017.4±145.9 pA (n=8) and −1705.9±109.0 pA (n=14, P<0.01 versus Ntg) in Ntg and Tg groups, respectively. The ≈70% increase in whole-cell ICa cannot be fully accounted for by the ≈20% increment in cell capacitance (Ntg 124.1±10.7 pF, n=19 and Tg 151.6±7.4 pF, n=26, P<0.05). Over the entire physiological voltage range tested (−40 mV to +50 mV), the average ICa density-voltage relationships for Tg cells and Ntg cells are bell-shaped, peak at 0 mV, and exhibit similar voltage-dependence. At 0 mV, the ICa density is elevated by 48% in Tg cells compared with the Ntg group. Thus, overexpression of the α1 subunit of LCC significantly increases the ICa density.
Previous studies indicate that the auxiliary subunits of the LCC, among other effects, modify the gating kinetics of the α1 subunit (eg, open time and inactivation rate) in an oocyte expression system.18 It is thus important to determine whether the kinetics of ICa in Tg myocytes differ from that in Ntg myocytes. Quantitative analysis revealed that decay of ICa in either group could be fitted to 2-exponential functions. Neither the fast (τfast) nor the slow decay time constant (τslow) in Tg cells differs from those in Ntg cells (Figure 1C). The relative magnitude of 2 components (Afast/Aslow) was also highly comparable (at 0 mV, 0.64±0.09, n=7 in Ntg and 0.70±0.19, n=9 in Tg, P=0.79). Thus, unlike other exogenous expression and reconstitution systems, overexpression of the α1 subunits in Tg mice produced LCCs that are indistinguishable from the native LCCs, in terms of kinetic properties of macroscopic ICa.
Unitary Currents of LCC in Ntg and Tg Cells
To test whether unitary properties of LCC are altered in Tg cells, we conducted a single-channel analysis of LCC currents using cell-attached patch-clamp technique with 5 mmol/L Ba2+ as the charge carrier. The representative patch from a Ntg myocyte (Figure 2A) contained 1 channel, and the patch from a Tg myocyte (Figure 2B) had at least 3 channels. The detected amplitude levels were plotted as amplitude frequency histograms (Figures 2C and 2D). A comparison of the amplitudes of the unitary currents (0.35 pA in Ntg, versus 0.39 pA for Tg cells) showed no significant difference (Figures 2C and 2D).
A linear regression of the voltage-dependence of single-channel current amplitude in the range of voltages used yielded a slope conductance of 10±1 pS for both Ntg and Tg groups (Figure 3A). Similarly, the individual conductances for each patch (11±4 pS for Ntg versus 10±3 pS for Tg cells, P>0.6) show no significant overexpression-induced changes in single-channel conductance. This relationship was unchanged by hyperpolarizing the holding potential to −80 mV (data not shown). Figure 3B describes the channel density on the surface membrane, including successful seals where no channel activity was observed, and in active patches alone. There is a significant increase in the number of channels per unit area in Tg cells. There is no significant change in open probability (Po, corrected for number of channels) of single LCC (P>0.4) due to overexpression of the α1 protein (Figure 3C).
Augmented Ca2+ Transients in the Tg Cardiac Myocytes
We next sought to determine whether the overexpressed LCCs can trigger additional Ca2+ release from the SR, and if so, whether the efficiency of LCC-to-RyR coupling becomes altered. The resting fluo-3 fluorescence intensity (F0) was 22.65±2.6 and 22.42±2.2 (in arbitrary unit) in Ntg (n=8) and Tg (n=14) cells, respectively, suggesting similar resting cytosolic free Ca2+ in both groups. Compared with Ntg cells, depolarization-elicited Ca2+ transients in Tg cells were uniformly enhanced throughout the cell and over all test voltages (Figure 4). This is corroborated by the statistics of systolic Δ[Ca2+] as a function of voltage (Figure 5A): the entire curve for the Tg cell is shifted upward, yet in both Tg and Ntg groups, the Δ[Ca2+] displays a similar bell-shaped voltage-dependence, which mirrors the voltage-dependence of the corresponding ICa (Figure 5B). This indicates that the overexpressed LCCs form functional coupling with the RyRs, triggering additional SR Ca2+ release in a graded fashion.
Coupling Efficiency Between LCCs and RyRs
The “gain” of EC coupling is quantified as the ratio of peak Δ[Ca2+] over the corresponding peak ICa, which reflects the efficiency of crosstalk between LCCs and RyRs. The gain in the Ntg group is monotonically decreased as a function of membrane potential, giving rise to an L-shaped voltage dependence (Figure 5C). This is in agreement with previous reports.16,23,24⇓⇓ In the Tg cells, the gain curve essentially overlaps with that of the Ntg in the negative voltage range, indicating that overexpressed LCCs are as effective as the native LCCs in triggering SR Ca2+ release. Given the exquisite spatial arrangement required for an efficient coupling of LCCs to RyRs,9,25⇓ this suggests a colocalization of overexpressed LCCs with native RyRs. At positive voltages, the curve for Tg cells bends upward and deviates significantly from that obtained from the Ntg cells (Figure 5C). The gain values in Tg cells at +20, +30, +40, and +50 mV are, respectively, 71%, 94%, 98%, 96% higher than those in Ntg cells (Figure 5C). This could be explained by an increased reverse mode NCX activity and/or enhanced cooperativity among LCCs in triggering SR Ca2+ release in Tg cells at high voltages (see Discussion).
Rate of Ca2+ Removal From the Cytosol in Voltage-Clamped Cells
To further understand the remodeling of Ca2+ regulation in the Tg heart, we examined kinetics of spatially averaged Ca2+ transients. A trend for a faster onset of Ca2+ transients (ie, smaller time to peak) was consistently observed at all test voltages in the Tg cells (data not shown), suggesting that the enhanced ICa may accelerate and synchronize the activation of SR Ca2+ release. More importantly, the decay of Ca2+ transient after repolarization was significantly hastened by ≈35% in Tg cells (Figure 5D). The magnitude of this change is comparable to that induced by an optimal β-adrenergic stimulation in the heart.26 This suggests that Ca2+ removal ability is substantially increased in response to the enhanced SL Ca2+ entry, perhaps as a compensatory mechanism to maintain Ca2+ homeostasis.
Action Potential- and Caffeine-Elicited Ca2+ Transients
In order to link the observations in voltage-clamped cells to in vivo EC coupling elicited by action potentials, we measured Ca2+ transients in myocytes under steady state electrical pacing (1 Hz, 1 minute). We detected an enhancement in systolic Ca2+ (Figure 6B) and no change in diastolic Ca2+ (F0=16.8±1.2, n=26 cells from 4 Ntg hearts; 16.7±1.0, n=24 cells from 4 Tg hearts). In contrast to Ca2+ transient decay under voltage-clamped conditions, we failed to detect any change in relaxation kinetics for action potential-elicited Ca2+ transients (Figure 6C) (see Discussion).
Next, the SR Ca2+ content was assessed by caffeine-induced Ca2+ transient. In both resting and electrically paced cells, there were no significant differences in caffeine-sensitive SR Ca2+ stores between Tg and Ntg groups (Figure 6D), in spite of enhanced SL Ca2+ entry. In the presence of caffeine, SR Ca2+ accumulation is blocked due to the leakage through RyRs, and the decay of Ca2+ transient mainly reflects the Ca2+ extrusion by NCX.10 We found that the T50 of caffeine-induced Ca2+ transients (2322±122 ms, n=26 Ntg cells) is, on average, 13-fold slower than the action potential-elicited Ca2+ transients (178±8 ms, n=26 Ntg cells). This observation suggests that the NCX contributes about 7% of the Ca2+ removal capability in this species, in agreement with a previous report.10 Interestingly, we found that the T50 of caffeine-induced Ca2+ transients is significantly reduced by about 27% in Tg versus Ntg cells (Figure 6E), suggesting an upregulation of the NCX activity in response to LCC overexpression.
Properties of Spontaneous Ca2+ Sparks
The cardiac Ca2+ transient in toto results from the summation of elemental SR Ca2+ release events, namely Ca2+ sparks originating from a few RyRs. Whether Ca2+ sparks become altered in cardiac hypertrophy remains controversial and appears to be model- and stage-dependent.15,27⇓ To gain a molecular perspective into altered Ca2+ signaling in the present murine model, we examined the properties of spontaneous Ca2+ sparks in resting Ntg and Tg myocytes. The rates of occurrence of Ca2+ sparks in Tg and Ntg cells are similar (Figure 7), suggesting that the genesis of spontaneous Ca2+ sparks is independent of LCC density. Parametric measurements of sparks further revealed that the unitary properties of the Ca2+ sparks, including amplitude, spatial width, and temporal duration, all remain unchanged (Figure 7). These data suggest that both the RyR Ca2+ sensitivity and the organization of release unit are unaltered in Tg cells. Moreover, the greater Ca2+ transients in Tg cells (Figures 5 and 6⇑) arise from a greater number of Ca2+ sparks activated per volume in Tg cells.
Expression of Ca2+ Cycling Proteins
To further probe the molecular mechanisms underlying the altered Ca2+ signaling in Tg cells, we analyzed the expression of major SL and SR Ca2+ cycling proteins in freshly prepared cardiac homogenates from Ntg and Tg mouse ventricles. Typical Western blots for RyR, NCX, SERCA2, and PLB are shown in Figure 8A, and the quantitative results are shown in Figure 8B. We detected a 53% increase in NCX protein, but expression levels of RyR, SERCA2, and PLB, key molecules in determining cardiac relaxation, were unchanged (Figure 8B).
Remodeling of Ca2+ Signaling in Tg Myocytes
In the present study, we provide a systematic quantification of intracellular Ca2+ signaling in a mouse in which a sustained and modest increase in cardiac Ca2+ was induced by an overexpression of the α1 subunit of the LCC.2,19⇓ At the 4-month period only a modest level of hypertrophy was observed.14 Electrophysiological recordings at both whole-cell and single-channel levels revealed that functional LCCs are overexpressed, with no detectable change in single-channel properties. Because ICa is also significantly elevated at an early age (2-months old), when the heart does not manifest any hypertrophy,2,19⇓ the increase in ICa should be considered as the cause, rather than a consequence of, cardiac morphological remodeling.
A major goal of this study is to gain insight into adaptation mechanisms that maintain Ca2+ balance and intracellular Ca2+ homeostasis in response to the constitutive enhancement of SL Ca2+ entry. We found that, at the microscopic level, properties of Ca2+ sparks are nearly identical regardless of LCC overexpression. Given that the SR Ca2+ content and resting Ca2+ are unaltered, this implicates a similar organization of the SR Ca2+ release units underlying spark generation in Tg and Ntg cells. At the macroscopic level, however, the amplitude of depolarization-elicited Ca2+ transients was increased. This suggests that LCC overexpression results in activation of a greater number of Ca2+ sparks (per volume) as well as greater fractional release of the SR Ca2+. Furthermore, in voltage-clamped cells, the increased ICa is amplified by the SR Ca2+ release proportionally (when Vm<+20 mV) or supraproportionally (when Vm≥+20 mV) in TG than Ntg cells. Therefore, the most prominent effect of transgenic overexpression of LCCs is an augmentation of systolic Ca2+ due to enhanced SL and SR Ca2+ cycling, whereas most Ca2+ homeostatic parameters remained unchanged after compensation, indicating an intrinsic plasticity of Ca2+ handling in the heart.
Importantly, we have identified an upregulation of the SL NCX, the primary route for SL Ca2+ extrusion, evidenced by a 53% and 27% increase in the expression and activity of NCX, respectively, in Tg myocytes. Further, the Ca2+ transient decay after repolarization was hastened by ≈35% in Tg versus Ntg cells. Because NCX normally contributes less than one tenth of the Ca2+ removal power (Figure 6), NCX upregulation should contribute only a small fraction to this relaxant effect. This suggests that the SR Ca2+ reuptake is augmented to a similar extent, which competes with the upregulated NCX for the common pool of cytosolic Ca2+ to maintain a proper SR Ca2+ load. Taken together, these results suggest that SL Ca2+ entry and extrusion, SR Ca2+ release and reuptake are altered in a coordinated fashion in Tg cells. The balanced Ca2+ influxes and effluxes across both SL and SR membranes help to explain the lack of an impact of LCC overexpression on many Ca2+ homeostatic parameters examined (resting and diastolic Ca2+, spontaneous Ca2+ sparks, and SR Ca2+ content), in spite of an augmented systolic Ca2+ following excitation.
The enhanced inward currents due to greater ICa and the NCX might be expected to prolong the action potential duration (APD) in Tg cells, although compensatory changes in potassium currents might normalize the APD. More work on these aspects is needed. The absence of a difference in the kinetics of Ca2+ transients in non-voltage-clamped cells is not unexpected because Ca2+ release prolongation may negate the relaxant effect of the enhanced Ca2+ removal ability in Tg hearts. Consequentially, both the peak and time-averaged Ca2+ levels in Tg cells would be significantly elevated during a cardiac cycle. It is perhaps the cumulative effect of this modest, yet sustained, imbalance of Ca2+ signaling that initiates and sustains the chain of signaling events that eventually lead to the morphological alterations observed in the Tg heart.2,14⇓
Orthograde and Retrograde Ca2+ Signaling Between LCCs and RyRs
The local control theory of cardiac EC coupling predicts that, for efficient Ca2+ coupling between LCC and RyR, these 2 Ca2+ channels have to colocalize to the 12-nm junctional clefts formed by the abutting SL and SR membranes.9,25⇓ Within the junctional cleft, Ca2+ emitted from RyRs provides a retrograde feedback signal that accelerates LCC inactivation.28,29⇓ This is manifested by the fact that abolition of SR Ca2+ release by caffeine increases the τfast of ICa decay from ≈7 to ≈16 ms.30 However, despite the overexpressed LCCs, neither the τfast nor its relative proportion is altered. This suggests that both overexpressed and native LCCs are sorted to the dyadic junctions in the same way, such that the Ca2+ signaling between LCCs and RyRs remains intact in both orthograde and retrograde directions in Tg myocytes.
Although ICa is the dominant Ca2+ source triggering EC coupling in the heart, Ca2+ influx via the reverse mode NCX may also evoke additional SR Ca2+ release, particularly at high voltages.10 In this respect, the curve of gain function in Tg, but not Ntg, cells bends upward at positive voltages, suggestive of SR Ca2+ release triggered by reverse-mode NCX in the Tg heart. Alternatively, it is also conceivable that LCC overexpression may permit a cooperative triggering of SR Ca2+ release by LCCs at the junction cleft, when LCC open probability is high. A quantitative appraisal for the relative contributions by these mechanisms merits future investigation.
It has been suggested that a slight disarray between RyRs and LCCs may occur in the hypertrophic and failing cardiac myocytes from salt-sensitive Dahl rats or spontaneous hypertensive and heart failure rats and account for the reduction of the LCC-to-RyR coupling efficiency.27 In a computer model of EC coupling, a mere 15-nm enlargement of the junction cleft reduces the systolic Ca2+ transient amplitude by two thirds, and dramatically desynchronizes the temporal pattern of the SR Ca2+ release.25 At the onset of cardiac hypertrophy in the present Tg model, we detected no reduction in the gain function of EC coupling and an apparently more synchronized SR Ca2+ release. These data suggest that manifestation of a compromised EC coupling appear to be a model- and stage-dependent phenomenon, as proposed by Balke and colleagues.15
In summary, a modest chronic increase in SL Ca2+ entry due to LCC overexpression induces a secondary upregulation of NCX activity and an enhancement of SR Ca2+ release and reuptake. However, the Ca2+ signaling efficiency between LCCs and RyRs is uncompromised. As a result, the systolic Ca2+ levels are augmented although most homeostatic parameters are able to retain their physiological setting points. These illustrate coordinated regulation and intrinsic plasticity of Ca2+ handling system in the heart.
This work was supported by NIH intramural grants (to R.-P.X., E.G.L., H.C.) and by NIH extramural grants P01 HL22619 (to A.S.), and T32 HL 07382 (J.N.M., M.R., and A.S.). The authors would like to thank Bruce Ziman for his excellent work on mouse cell isolation.
↵*Both authors contributed equally to this work.
Original received July 3, 2001; resubmission received November 1, 2001; accepted November 21, 2001.
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