Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

Abstract

BACKGROUND:

Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca²⁺-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca²⁺ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart.

OBJECTIVE:

To explore whether enhanced Ca²⁺ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca²⁺ (I_Ca-L) currents.

METHODS:

The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals.

RESULTS:

The amplitudes of I_Ca-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I_Ca-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I_Ca-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca²⁺ with BAPTA.

CONCLUSIONS:

These findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L currents. Modified I_Ca-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.

Calcium ion (Ca²⁺) homeostasis in cardiac myocytes is critical for normal mechanical function of the heart. Voltage-operated L-type Ca²⁺ (I_Ca-L) channels in the plasma membrane of cardiomyocytes constitute a major influx pathway for extracellular Ca²⁺ (1–3). Ca²⁺ entry through I_Ca-L channels triggers the efflux of Ca²⁺ from the sarcoplasmic reticulum (SR) via SR Ca²⁺ release channels, which are also referred to as ryanodine receptors (4,5). Additional Ca²⁺ cycling proteins include the Na⁺/Ca²⁺ exchanger and the Ca²⁺-ATPase in the sarcolemma as well as the SR Ca²⁺-ATPase SERCA2a (1,6,7). These Ca²⁺ transport mechanisms are essential for the homeostasis of basal Ca²⁺ concentration inside the cardiomyocyte and the dynamic Ca²⁺ oscillations during the contraction-relaxation cycle of the heart (1,8).

Our earlier study (9) indicated that transgenic (TG) overexpression of SERCA2a in rat hearts enhanced Ca²⁺ uptake into the SR. Thus, isometrically contracting papillary muscles from SERCA2a TG rats had increased contraction amplitudes and elevated contraction-relaxation rates compared with preparations from non-TG (NTG) rats (9). Consistent with an increased SR Ca²⁺ cycling capacity, the myocardium of TG rats responded with stronger positive inotropic and lusitropic effects to forskolin-induced adenylate cyclase activation than myocardial tissue of NTG rats (9). These findings, together with the reports from another study (10), suggested a more rapid re-uptake of Ca²⁺ into the cardiac SR of SERCA2a TG rats. Consequently, increased Ca²⁺ loading of the SR (11) may enhance Ca²⁺ release during contractions and thereby improve the mechanical performance of the hearts of SERCA2a TG rats under normal and pathophysiological conditions.

Although TG expression of SERCA2a in the heart has already been used to restore impaired SR Ca²⁺ handling in failing hearts (12–16), the molecular mechanisms underlying this beneficial effect are not completely understood. In particular, it is unknown whether forced expression of SERCA2a and, hence, accelerated re-uptake of Ca²⁺ into the SR, also modulates other Ca²⁺ influx pathways in cardiomyocytes (ie, I_Ca-L channels and the Na⁺/Ca²⁺ exchanger) (1). Thus, it is conceivable that elevated SERCA2a interferes with the feedback control of transmembrane I_Ca-L currents in cardiac myocytes, which is exerted by Ca²⁺ through calmodulin-dependent pathways (17–19). Furthermore, TG expression of SERCA2a can possibly modify the positive inotropic effect of sympathetic nerve activation through changes in I_Ca-L currents.

The goal of the present study was to assess whether improved Ca²⁺ cycling in the hearts of SERCA2a TG rats is associated with changes in I_Ca-L currents. Therefore, we compared the kinetics of I_Ca-L in single cardiomyocytes of normal and SERCA2a TG rats. The patch-clamp technique was used to measure whole-cell currents in freshly isolated cardiomyocytes from the hearts of SERCA2a TG rats and from age-matched wild-type (ie, NTG) animals. Rats with TG expression of SERCA2a were generated in our laboratory, and their myocardial contractile function was described in detail previously (9). To challenge intracellular Ca²⁺ handling and to mimic sympathetic nerve activation, we also analyzed the effect of the beta-adrenergic agonist isoproterenol (Iso) on I_Ca-L in cardiomyocytes from NTG and SERCA2a TG rats.

METHODS

Animals

The present investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (Publication No. 85-23, revised 1996). Experiments were performed with the hearts of 20-weekold male NTG rats (n=34) and 38 TG rats overexpressing rat SERCA2a. The total number of recorded cells was 49 and 53 in NTG and TG hearts, respectively. Transgene expression was under control of the human cytomegalovirus immediate early enhancer linked to the chicken beta-actin promoter. A detailed description of the generation of SERCA2a TG rats and the contractile function of their hearts is given elsewhere (9). The animals were anesthetized with ether and sacrificed by decapitation.

Cell diameter determination

Left ventricular tissue samples of NTG and TG rats were embedded in paraffin, cut into 3 μm sections and subjected to hematoxylin-eosin staining. Three different stained sections of NTG and TG (n=6 each) tissue were morphometrically analyzed to quantify the diameter of the myocytes (10 myocytes per section) using a video camera equipped with a video control system and connected to a Zeiss Axiophot microscope (Carl Zeiss, Germany). Image analysis was performed using Scion Image software version 1.62a (Scion Co, USA) on a Power Macintosh 8200/120 computer (Apple Computer Inc, USA).

Western blotting

Solubilized homogenates of left ventricular protein (20 μg per lane) were separated by sodium dodecyl polyacrylamide gel electrophoresis (T=13.8%, C=3.2%) as described in detail elsewhere (9). Protein transfer to polyvinylidene fluoride membranes (Roche, Germany), membrane blocking, and detection and quantification of SERCA2a and phospholamban with specific monoclonal antibodies (1:1000 dilution of clone 2A7-A1 for SERCA2a [Affinity BioReagents, USA] and 1:2000 dilution of clone A1 for phospholamban [Millipore, USA]) were similarly performed as reported previously (9). The signal from the horseradish peroxidase-conjugated affinity-purified goat anti-mouse immunoglobulin ([H+L] IgG, Dianova, USA) used as secondary antibody was developed using Super Signal West Pico substrate (Thermo Scientific, USA). The signals were collected in a Fusion FX7 detection system (PEQLAB Biotechnology GmbH) and quantified by densitometry analysis using Bio-1D version 12.12 (PEQLAB Biotechnology GmbH, Germany). The optical densities determined were considered to reflect the relative amounts of the detected proteins. In some experiments, distinct amounts of protein per lane were used to check the linearity of the reaction.

Isolation of single myocytes from rat ventricular myocardium

Ventricular cardiac myocytes were dispersed using a standard collagenase dissociation protocol as described in detail elsewhere (20,21). Briefly, the hearts were quickly excised and the aorta was cannulated in a retrograde manner. The hearts were perfused at a constant flow rate of 7 mL/min with a Ca²⁺-free physiological salt solution in a nonrecirculating Langendorff apparatus using a roller pump. After an initial perfusion period of 5 min to flush the coronary vessel system of blood, the hearts were perfused for 30 min with a Ca²⁺-free physiological salt solution supplemented with 0.1% collagenase (type II, Worthington, USA) and 20 μM CaCl₂. The perfusate was equilibrated with 95% O₂ and 5% CO₂ (pH 7.4) at 37°C. Finally, the ventricles were chopped and gently triturated to release the cells into Kraftbruehe medium. The cell suspension was filtered, resuspended and kept in Kraftbruehe medium at room temperature for at least 2 h.

Electrophysiological measurements

Membrane potentials and whole-cell currents were recorded with an EPC-7 amplifier (Heka Elektronik, Germany) connected to a personal computer via interface. The data were filtered at 2 kHz, sampled at 5 kHz and evaluated using custom written software. Depolarizing pulses from −45 mV to 0 mV with a duration of 350 ms were applied at a frequency of 1 Hz. Current flow through I_Ca-L channels was calculated as the difference between the negative peak current and the late current at the end of the pulse. The measurements were performed for approximately 30 min with stable access resistance and capacitive current. The current-voltage relations were obtained by 20 pulses of 140 ms or 350 ms duration (1 Hz). Starting from a holding potential of −45 mV, the membrane potential was adjusted stepwise to values of between −50 mV and +50 mV.

Reagents and solutions

During the voltage-clamp experiments, ventricular cardiomyocytes were superfused at 37°C with a bath solution containing 150 mM NaCl, 5.4 mM CsCl, 1.8 mM CaCl₂, 1.2 mM MgCl₂, 20 mM glucose and 5 mM HEPES, titrated to pH 7.4 with NaOH. Whole-cell, patch-clamp recordings were performed using patch pipettes (1.8 MΩ tip resistance) filled with Cs⁺ electrode solution that contained 140 mM CsCl, 5 mM Na₂ATP, 5.5 mM MgCl₂, 10 mM HEPES and 0.01 mM EGTA, titrated to pH 7.2 with CsOH. K⁺ channel currents were suppressed by substituting Cs⁺ for K⁺ in both the bath and the electrode solutions. In some experiments, the electrode solutions were filled additionally with 5 mM BAPTA (Sigma-Aldrich, USA) to adjust the intracellular Ca²⁺ concentration to approximately 10 nM. All other compounds were added to the bath solution at the indicated final concentrations.

Data presentation and statistical analysis

The data were sampled and fitted according to the following two-exponential equation:

$$\text{Y}={\text{Y}}_0+{\text{A}}_1{e}^{-[(\text{x}-{\text{x}}_0)/{\tau }_{\text{fast}}]}+{\text{A}}_2{e}^{-[(\text{x}-{\text{x}}_0)/{\tau }_{\text{slow}}]}$$

Data for repeated measurements were analyzed using two-way ANOVA followed by a post hoc Bonferroni test to calculate statistical significance of single data points with Prism 3.0 software (GraphPad Software Inc, USA).

RESULTS

Physical characteristics of SERCA2a TG and NTG rats are summarized in Table 1. No significant differences were observed between TG and NTG rats. In particular, the left ventricular weight to body weight ratios and the cardiomyocyte diameters were not significantly different between the two groups. Protein expression of SERCA2a and phospholamban in the hearts of TG and NTG rats were assessed by semiquantitative Western blot analysis using specific antibodies. SERCA2a protein levels were significantly higher in TG rats (4.5±0.2 versus 3.4±0.2 arbitrary units; P<0.05), but no significant differences in phospholamban were detected between TG and NTG rats (Figure 1).

TABLE 1.

Characteristics of nontransgenic (NTG) and SERCA2a transgenic (TG) rats

Parameter	NTG (n=6)	TG (n=6)
BW, g	416.7±9.2	425.0±9.6
Heart weight, mg	1330.7±48.9	1355.0±42.0
LV, mg	946.8±33.8	943.0±34.4
LV/BW, mg/g	2.27±0.07	2.21±0.03
Myocyte diameter, μm	13.1±0.2	13.2±0.3

Data presented as mean ± SEM. The analyzed animals were littermates of animals used for enzymatic cardiomyocyte isolation. Differences between TG and NTG rats were not statistically significant. BW Body weight; LV Left ventricular weight

Figure 1).

Expression of the sarcoplasmic reticulum proteins SERCA2a and phospholamban in the ventricular myocardium of SERCA2a transgenic (TG) and nontransgenic (NTG) rats. Representative Western blots for SERCA2a and monomeric phospholamban (PLB_low) from two different animals each, TG and NTG, are shown. Note: SERCA2a protein levels were significantly higher in TG than in NTG rats (4.5±0.2 arbitrary units versus 3.4±0.2 arbitrary units [n=6 each]; P<0.05). No significant differences in PLB_low expression between TG and NTG rats were detected

I_Ca-L currents are enhanced in cardiomyocytes of SERCA2a TG rats

Whole-cell patch clamp recordings were performed to measure transmembrane I_Ca-L currents in freshly isolated cardiomyocytes of TG rats overexpressing cardiac SERCA2a and NTG animals. Representative original current curves are shown in Figure 2A. Fitted inactivation traces of I_Ca-L currents obtained by normalizing the original data of NTG and TG to a two-exponential equation are depicted in Figure 2B. The average peak I_Ca-L at 0 mV was −1612±55 pA (18 recorded cells) and −1985±48 pA (21 recorded cells) in NTG and TG rats, respectively (P<0.01). Quantitative analysis of the decay of I_Ca-L at the voltage producing peak inward current revealed significant differences in the inactivation characteristics of I_Ca-L in cardiomyocytes of TG and NTG rats. Compared with NTG cardiomyocytes, the tau fast (τ_fast) and tau slow (τ_slow) decay time constants in TG cardiomyocytes were increased by 38% and 16%, respectively (Figure 3). Over the entire voltage range tested (−50 mV to +50 mV), the average I_Ca-L voltage relationship for TG and NTG cardiomyocytes was bell-shaped, peaked at 0 mV and exhibited a similar voltage dependence (Figures 4B and 4E). Thus, compared with NTG cells, isolated cardiomyocytes of rats overexpressing cardiac SERCA2a showed differences in the magnitude and inactivation kinetics of I_Ca-L under basal SR Ca²⁺ cycling conditions.

Figure 2).

Whole-cell patch-clamp recordings of L-type Ca²⁺ (I_Ca-L) currents in ventricular cardiomyocytes of wild-type rats (ie, nontransgenic [NTG]) and of transgenic (TG) rats overexpressing SERCA2a. A Representative original registration of a typical whole-cell I_Ca-L current activated by membrane depolarization from −45 mV to 0 mV for 140 ms. Note: I_Ca-L current amplitudes were significantly higher in the cardiomyocytes of TG than NTG hearts throughout all experiments. B Normalized inactivation curves of I_Ca-L. The currents were fitted with a two-exponential equation. Inactivation of I_Ca-L channels was clearly slower at the voltage-producing peak inward current in SERCA2a TG than in NTG cells

Figure 3).

Effect of isoproterenol (Iso, 50 nM) on peak current amplitudes and time constants (tau fast and tau slow) of the decay of I_Ca-L at 0 mV in cardiomyocytes of nontransgenic (NTG) and transgenic (TG) rats overexpressing SERCA2a. Experiments were performed in the absence and presence of the Ca²⁺ chelator BAPTA (5 mM). Values presented as mean ± SEM. *P<0.05 between vehicle control (Con) and isoproterenol (Iso); ^†P<0.05 for TG versus NTG. Data were obtained by analysis of fitted curves from respective original current traces as described under Methods. Data in panels A, C and E were calculated from 18 and 21 single cells of 10 NTG and 12 TG animals, respectively. Data in panels B, D and F were obtained from 12 and 11 single cells of seven NTG and six TG animals, respectively

Figure 4).

Effect of isoproterenol (Iso) on whole-cell L-type Ca²⁺ (I_Ca-L) currents in cardiomyocytes of wild-type (ie, nontransgenic [NTG]) and SERCA2a transgenic (TG) rats. The effect of Iso (50 nM) compared with superfusion without the beta-agonist (Con) is depicted in panels A and D. Note: application of Iso increased the I_Ca-L amplitudes more in the cardiomyocytes of TG than in NTG rats. Current-voltage relationships in NTG (B) and SERCA2a TG cardiomyocytes (E) in the absence (square symbols) and presence of 10 nM (circles) or 50 nM Iso (triangles). Effect of Iso (10 nM, 50 nM) on the time course of peak I_Ca-L current in normal (C) and cardiomyocytes overexpressing SERCA2a (F)

I_Ca-L in cardiomyocytes of SERCA2a TG rats are sensitive to beta-adrenergic stimulation

To determine whether the observed differences in I_Ca-L currents under resting Ca²⁺ cycling conditions were further enhanced during forced Ca²⁺ handling, the effect of beta-adrenergic stimulation with Iso in isolated cardiomyocytes of TG and NTG rats was studied. Iso stimulates Ca²⁺ cycling in cardiomyocytes by activating several protein kinase A-dependent Ca²⁺ pathways including sarcolemmal Ca²⁺ influx via I_Ca-L channels (22,23) and phospholamban-regulated SERCA2amediated transport of Ca²⁺ into the SR (24). Representative tracings of I_Ca-L currents from cardiomyocytes of NTG and TG animals, and the changes in I_Ca-L amplitudes in response to Iso (50 nM) are shown in Figure 4. As predicted, beta-adrenergic stimulation increased the peak I_Ca-L amplitudes at voltage values of between −40 mV and +50 mV in a dose-dependent and reversible manner in cardiac myocytes of TG and NTG rats. The effect of Iso on I_Ca-L amplitudes was markedly enhanced in TG compared with NTG cardiomyocytes (3.2-fold increase from control values versus 2.3-fold in NTG; P<0.001).

The typical effect of Iso on the inactivation kinetics of I_Ca-L in TG and NTG cardiomyocytes is shown in Figure 3. At the voltage producing peak inward current, quantitative analysis of the decay of I_Ca-L revealed an accelerated inactivation rate by Iso in TG (τ_fast 7.4±0.8 ms versus 13.5±0.3 ms without Iso; τ_slow 61.3±2.4 ms versus 74.3±1.1 ms without Iso) but not in NTG rats (τ_fast 8.9±0.5 ms versus 9.8±0.6 ms without Iso; τ_slow 60.3±1.2 ms versus 64.0±1.5 ms without Iso, [Figure 3]). Thus, the differences in the inactivation kinetics of I_Ca-L between TG and NTG cardiomyocytes under basal conditions were abolished by Iso during forced Ca²⁺ handling (Figures 3 and 5).

Figure 5).

Original registration of the typical whole-cell inactivation of L-type Ca²⁺ (I_Ca-L) channels in cardiomyocytes of wild-type (ie, nontransgenic [NTG]) (A) and SERCA2a transgenic (TG) (B) rats in the absence (control [Con]) and presence of 50 nM isoproterenol (Iso). Fitted normalized inactivation curves of I_Ca-L from wild-type and TG hearts overexpressing SERCA2a are shown in panels C and D, respectively. Inactivation of I_Ca-L was clearly faster at the voltage-producing peak inward current after addition of Iso in both NTG and TG cardiomyocytes (P<0.0001). Fitted normalized inactivation curves of I_Ca-L in NTG and TG cardiomyocytes after application of 50 nM Iso are shown in panel E. Time constants for inactivation of I_Ca-L were not significantly different between NTG and TG during beta-adrenergic stimulation with Iso

Changes in I_Ca-L currents in cardiomyocytes from SERCA2a TG rats are Ca²⁺ dependent

Increased cytosolic Ca²⁺ resulting from entry of extracellular Ca²⁺ via I_Ca-L channels and/or Ca²⁺ release from the SR is part of a negative feedback loop that modulates I_Ca-L through calmodulin kinase II (CaMKII)-dependent mechanisms (17,19). To determine whether the observed differences in the inactivation kinetics of I_Ca-L between TG and NTG cardiomyocytes were Ca²⁺ dependent, whole-cell, patch-clamp I_Ca-L recordings were performed in single cardiomyocytes loaded with the rapid Ca²⁺ buffer BAPTA. An intracellular Ca²⁺ concentration of approximately 10 nM was calculated with a BAPTA concentration of 5 mM. As shown in Figure 3, no differences in peak amplitudes and inactivation kinetics of I_Ca-L were found between TG and NTG cardiomyocytes in the presence of BAPTA. Furthermore, Iso-stimulated average peak I_Ca-L currents at 0 mV were similar in BAPTA-loaded TG and NTG cardiomyocytes. In contrast, the inactivation rate of I_Ca-L in response to beta-adrenergic stimulation with 50 nM Iso was markedly faster in TG than in NTG cardiomyocytes during Ca²⁺ buffering with BAPTA. Hence, the differences in the amplitude and inactivation kinetics of I_Ca-L between TG and NTG cardiomyocytes were clearly Ca²⁺ dependent.

DISCUSSION

As the major novel finding of the present study, we reported substantial differences between whole-cell I_Ca-L currents of single cardiomyocytes in TG rats overexpressing SERCA2a and NTG animals. Differences included increased I_Ca-L current amplitudes and delayed inactivation kinetics of I_Ca-L in the cardiomyocytes of SERCA2a TG rats. Several lines of evidence suggest that these changes in I_Ca-L were related to improved Ca²⁺ cycling in the hearts of SERCA2a TG rats. Thus, increased I_Ca-L amplitudes are consistent with enhanced transmembrane Ca²⁺ influx through I_Ca-L channels in cardiomyocytes of SERCA2a TG rats. Consequently, Ca²⁺-induced release of Ca²⁺ ions from the SR, which is likely enhanced in cardiomyocytes with the SERCA2a transgene due to augmented Ca²⁺ loading, would further increase the cytosolic Ca²⁺ concentration. The rise in intracellular Ca²⁺, in turn, facilitates excitation-contraction coupling and may exert a positive inotropic effect in hearts overexpressing SERCA2a. It remains to be clarified whether the elevated I_Ca-L amplitudes in the cardiomyocytes of SERCA2a TG rats resulted from increased ion channel density in the plasma membrane, or whether they were due to a higher open probability of single I_Ca-L channels. The I_Ca-L amplitudes at the voltage producing peak inward current were particularly susceptible to beta-adrenergic stimulation in cardiac myocytes overexpressing SERCA2a. In keeping with this observation, pharmacological stimulation with the beta-adrenergic agonist Iso elicited stronger inotropic and lusitropic effects in isolated perfused hearts (data not shown) and in isolated papillary muscles from SERCA2a TG rats with streptozotocin-induced diabetes mellitus than in preparations from diabetic wild-type rats (9). Clinical studies (25) indicate that exercise-related left ventricular dysfunction in early diabetic cardiomyopathy involves a defective recruitment of myocardial contractility. The abnormal cardiac response to physical exercise was attributed to impaired sympathetic innervation of the diabetic heart (25,26). Thus, our findings raise the possibility that TG overexpression of SERCA2a can restore myocardial contractility in such diseased states through enhanced susceptibility of I_Ca-L to beta-adrenergic stimulation. On the other hand, improved Ca²⁺ reuptake into the SR of SERCA2a TG cardiomyocytes may accelerate relaxation and, thereby, partly compensate for the reduced intracellular SR Ca²⁺ transport capacity in diseases such as diabetic cardiomyopathy.

In addition to the increased I_Ca-L amplitudes, the inactivation kinetics of I_Ca-L currents between cardiomyocytes of NTG and SERCA2a TG rats were clearly different. The general consensus is that inactivation of I_Ca-L during membrane depolarization of cardiac myocytes is determined by voltage-and Ca²⁺-dependent mechanisms (1,27–29). Two phases in Ca²⁺-dependent inactivation of I_Ca-L currents can normally be distinguished: a slower component, which is related to transmembrane influx of Ca²⁺ from extracellular space, and a fast component that depends on Ca²⁺ release from the SR (1,27,29). We hypothesize that the different inactivation kinetics of I_Ca-L channels adjust the temporal precision of the intracellular Ca²⁺ signal and thereby contribute to the improved contractile performance of the hearts of SERCA2a TG rats.

Consistent with the results of previous studies (30,31), beta-adrenergic stimulation of I_Ca-L was sensitive to intracellular Ca²⁺ buffering with BAPTA. Likewise, depletion of intracellular Ca²⁺ stores with ryanodine and SERCA2a inhibition with thapsigargin prevented the stimulatory effect of Iso on I_Ca-L amplitudes in cardiomyocytes of NTG and TG rats (data not shown). Ryanodine and thapsigargin also delayed the fast inactivation component of I_Ca-L in both groups (data not shown). These observations are in agreement with a model of use-dependent facilitation of I_Ca-L in cardiac myocytes. According to this model, an increase in the amplitude and a slowing of inactivation of I_Ca-L occurs during a sudden rise in heart rate (32–37). Recent investigations (38) revealed that the negative feedback control of I_Ca-L, which is exerted by Ca²⁺ released from the SR, represents a critical mechanism during facilitation. Depletion of intracellular Ca²⁺ is, therefore, expected to prolong the inactivation of I_Ca-L. A role for Ca²⁺ in the negative feedback control of I_Ca-L channels is also supported by our observation that the decay of I_Ca-L in response to Iso was accelerated in the cardiomyocytes of SERCA2a TG rats. Because beta-adrenergic stimulation activates SERCA2a via phosphorylation of phospholamban (24), Ca²⁺ release from the SR in response to Iso is presumably pronounced in the cardiomyocytes of SERCA2a TG rats. Accordingly, inactivation of I_Ca-L was clearly faster in cardiomyocytes of mice with targeted disruption of the gene encoding phospholamban (39).

SUMMARY

Our findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a TG rats, under basal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L current. These changes are Ca²⁺ dependent and possibly contribute, at least to some extent, to the improved contractile performance of the SERCA2a TG myocardium.