Rescue of Contractile Parameters and Myocyte Hypertrophy in Calsequestrin Overexpressing Myocardium by Phospholamban Ablation*

Yoji SatoDagger §, Helen KiriazisDagger , Atsuko YataniDagger , Albrecht G. SchmidtDagger , Harvey Hahn||, Donald G. Ferguson**, Hidenori SakoDagger , Sayaka MitaraiDagger , Ritsu HondaDagger , Laurence Mesnard-RouillerDagger , Konrad F. FrankDagger , Beate BeyermannDagger , Guangyu Wu||, Kannosuke Fujimori§, Gerald W. Dorn II||, and Evangelia G. KraniasDagger DaggerDagger

From the Dagger  Department of Pharmacology and Cell Biophysics, and || Division of Cardiology, University of Cincinnati, Cincinnati, Ohio 45267, the § Division of Xenobiotics, Metabolism and Disposition, National Institute of Health Sciences, Tokyo 158, Japan, and the ** Department of Anatomy, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, August 1, 2000, and in revised form, December 11, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca2+-induced Ca2+ release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca2+ storage, assessed by caffeine-induced Na+-Ca2+ exchanger currents. The inactivation time of the L-type Ca2+ current (ICa), which has an inverse correlation with Ca2+-induced SR Ca2+ release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of ICa to trigger SR Ca2+ release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha -skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypertrophy of ventricular myocardium is postulated to be an adaptive response to relative increases in external workload, induced by endocrine, paracrine, autocrine, and mechanical factors or decreased myocardial contractility (1). The increase in heart mass has been implicated to normalize cardiac function by decreasing wall stress. However, a sustained imbalance between workload and muscle contractility may lead to progressive thinning of the left ventricular wall and chamber dilation associated with decompensated hypertrophy and heart failure (2, 3). Studies in human and animal models have shown that cardiac hypertrophy is associated with impaired sarcoplasmic reticulum (SR)1 Ca2+ modulation, leading to aberrant cardiac contraction and relaxation (4-8). Although several Ca2+-related signaling molecules, such as calcineurin, Ca2+-calmodulin kinase, and Ca2+-dependent protein kinase C have been suggested to play key roles in myocardial hypertrophic responses (9-12), it is not clear yet whether the abnormal SR Ca2+ handling per se contributes to the generation, maintenance, and characteristics of cardiac hypertrophy in vivo. Furthermore, it is unknown whether reversal of the SR Ca2+ handling defects can mediate functional benefits in myocardial hypertrophy.

The SR plays a key role in the regulation of Ca2+ homeostasis and contractility in cardiac muscle. During relaxation, Ca2+ is transported from the cytosol into the lumen of the SR by a Ca2+-ATPase. Subsequently, in response to Ca2+ influx through L-type Ca2+ channels, the Ca2+ loaded in the SR is released through the ryanodine receptors for the initiation of muscle contraction. The activity of the SR Ca2+-ATPase is regulated by phospholamban, a 52-amino acid phosphoprotein (13). Decreases in the levels of phospholamban or increases in its phosphorylation status result in an increase in the apparent Ca2+ affinity of the SR Ca2+-ATPase, and augment cardiac contractile parameters. The mechanisms underlying these regulatory effects of phospholamban have been suggested to reflect facilitation of SR Ca2+ transport, Ca2+ loading, and Ca2+ release (13-19). These studies also indicate that the SR Ca2+ load is one of the major determinants of myocardial contractility (18, 19). The SR Ca2+ load and subsequent Ca2+ release are regulated by calsequestrin, a luminal SR protein, with high capacity and low affinity for Ca2+ (20). Calsequestrin has been reported to form a stable complex with the ryanodine receptor, junctin, and triadin (21). Recent transgenic approaches have shown that increased expression of calsequestrin in the heart was associated with increased SR Ca2+ storage capacity, but this SR Ca2+ was not available for release during excitation-contraction coupling, leading to depressed Ca2+ transients and contractile parameters (22, 23). The depressed cardiac function was associated with re-expression of a fetal gene program and hypertrophy (23) or failure (24). To better define the role of SR Ca2+ handling defects in the hypertrophic response, the current study employed a genetic approach to improve SR function through phospholamban inhibition in the calsequestrin overexpressing hearts (23). Ablation of phospholamban restored the depressed contractile parameters and reversed the compensatory alterations in SR Ca2+ handling protein levels. Furthermore, induction of atrial natriuretic factor (ANF) and alpha -skeletal actin was normalized, and myocyte hypertrophy was rescued in the calsequestrin overexpressing hearts.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experimental Animals-- Phospholamban knockout 129SvJ/CF-1 mice (15) were mated with transgenic FVB/N mice overexpressing murine cardiac calsequestrin specifically in cardiac muscle (line number 418) (23). F1 heterozygous phospholamban offsprings carrying the calsequestrin transgene were inbred with their littermates without the transgene to obtain F2 pups with three different genotypes: wild-type (WT), calsequestrin overexpressing (CSQOE), and calsequestrin overexpressing with phospholamban ablation (CSQOE/PLBKO). To identify the genotypes, polymerase chain reaction analysis of tail genomic DNA was carried out as described previously (15, 23). The cardiac phenotype of the WT mice with mixed genetic background was similar to that in the FVB/N genetic background (23). The handling and maintenance of animals in this study was approved by the ethics committee of the University of Cincinnati. Eight to 15-week-old mice of either gender were used for the following studies unless otherwise indicated.

Morphological Analyses-- Immunocytochemistry of cardiac ventricles were performed as described previously (23). Ventricular myocyte cross-sectional areas were obtained, as described previously (25).

In Vivo and ex Vivo Left Ventricular Function-- Left ventricular contractile parameters were determined in closed-chest anesthetized mice using a 1.4-French scale Millar MIKRO-TIP catheter, as previously described (25). Contractile parameters of isolated hearts were also determined in Langendorff mode at 37 °C with a constant perfusion pressure of 50 mm Hg, as described previously (23).

Electrophysiology of Isolated Cardiomyocytes-- Left ventricular myocytes were isolated from mice and whole cell currents were recorded using patch clamp techniques as described previously (17, 26, 27). Membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of -50 mV. Whole cell Na+-Ca2+ exchanger currents (INCX) were recorded by the method Kimura et al. (28). To activate Na+-Ca2+ exchanger, the cells were held at -40 mV and the external solution was rapidly switched to one in which equimolar LiCl was substituted for NaCl. The Ca2+ content of the SR was evaluated in voltage-clamped myocytes by the transient inward INCX currents evoked by a rapid application of caffeine (5 mM) to release the SR Ca2+ (29). Whole cell L-type Ca2+ currents (ICa) were recorded by applying depolarization pulses every 10 s from a holding potential of -50 mV (26, 27). To obtain relations between peak current density and inactivation kinetics, ICa was measured in a series of external Ca2+ concentrations (1.0-20 mM). Action potentials were recorded, as previously described (27).

Immunoblotting and Dot-blot Analysis-- SDS-polyacrylamide electrophoresis and quantitative immunoblotting of cardiac ventricular homogenates were performed as described previously (23). Dot-blot analysis of total RNA from cardiac ventricles was performed as described previously (25).

Statistics-- Data are presented as mean ± S.E. Comparisons across groups were evaluated using analysis of variance (ANOVA). When the p value was less than 0.05, Fisher's least significant difference (LSD) procedure was employed to discriminate which means differed from the others.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calsequestrin Overexpressing Mice Deficient in Phospholamban-- To determine whether enhanced SR Ca2+ transport activity may modify the time course of cardiac dysfunction and hypertrophy, we generated transgenic mice, which overexpress calsequestrin and are deficient in phospholamban. To achieve this, CSQOE mice, which exhibit depressed function and hypertrophy (23), were mated with phospholamban knockout mice, which demonstrated enhanced SR Ca2+ transport and hyperdynamic function (15). F2 littermates with WT, CSQOE, and CSQOE/PLBKO genotypes were characterized in parallel to eliminate any potential effects of the genetic background. There were no phenotypic alterations at the gross morphological level between the three models up to 7 months of age. Furthermore, ablation of phospholamban did not alter the levels of calsequestrin overexpression (Fig. 1A), which was increased by 18 ± 4 (n = 8) in CSQOE and 19 ± 4-fold (n = 8) in CSQOE/PLBKO ventricles, compared with WT littermates. Phospholamban was undetectable in CSQOE/PLBKO, whereas it was increased by 37% in CSQOE, compared with WTs, consistent with previous findings (23).


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Fig. 1.   Quantitative immunoblotting and immunolocalization studies confirming calsequestrin overexpression in the phospholamban knockout background. A, a dilution series of a control homogenate from pooled wild-type hearts was used to generate a standard line for quantitation of calsequestrin and phospholamban ventricular protein levels in WT, CSQOE, and CSQOE/PLBKO littermate mice. Calsequestrin levels were ~20-fold above WT in CSQOE and CSQOE/PLBKO. Phospholamban was increased in CSQOE (37%), but was absent in CSQOE/PLBKO. B, longitudinal sections of ventricular tissue. Images were obtained using identical brightness and contrast. Clearly calsequestrin-specific staining was much brighter in CSQOE and CSQOE/PLBKO. There were no obvious differences in staining patterns in any of the sections, suggesting that the overexpression of calsequestrin or the absence of phospholamban does not affect the distribution of calsequestrin. Bar, 8 µm.

Subcellular Distribution of Calsequestrin-- To assess the effects of phospholamban ablation on the subcellular localization of overexpressed calsequestrin, immunostaining and confocal microscopy were performed. Cryostat sections from both CSQOE and CSQOE/PLBKO transgenic hearts exhibited similar striated staining patterns as WTs (Fig. 1B). The increased staining in CSQOE and CSQOE/PLBKO tissue was consistent with the immunoblotting data. Further examination of sections from the CSQOE and CSQOE/PLBKO littermates revealed that the staining patterns of transverse striations were predominant. No obvious morphological deterioration, such as abnormal striations, fibrosis, and necrosis, was observed in these sections.

Rescue of Cardiac Contractile Parameters-- An important question is whether the depressed cardiac contractile parameters associated with calsequestrin overexpression may be restored in CSQOE/PLBKO mice. Thus, cardiac catheterization was performed in 2-month-old intact anesthetized animals and their hemodynamic function was evaluated. The intrinsic heart rate was not different between groups. However, the maximal rates of left ventricular pressure development (+dP/dt) and decline (-dP/dt), which were significantly attenuated in the CSQOE mice (6,677 ± 199 and 6,244 ± 546 mm Hg/s, respectively), exhibited values that were similar between CSQOE/PLBKO (10,807 ± 906 and 10,552 ± 942 mm Hg/s, respectively) and WT mice (10,333 ± 1,054 and 12,483 ± 1,363 mm Hg/s, respectively), indicating restoration of cardiac function in vivo.

These studies were extended to the organ level, and isolated mouse hearts from 2-month-old littermates were perfused in parallel, using the Langendorff mode (Fig. 2). Hearts from CSQOE mice exhibited depressed +dP/dt values, similar to previous observations in the CSQOE mouse of the FVB/N genetic background (23). However, the rates of left ventricular relaxation, which were attenuated in the original model (23), did not reveal any statistical differences from their WT littermates (FVB/N + 129SvJ/CF-1). This apparent discrepancy may be due to the differences in the genetic background (FVB/N versus FVB/N+129SvJ/CF-1), which is known to play a major role in determining the cardiac phenotype of mice (30, 31). Interestingly, the contractile parameters in CSQOE hearts did not deteriorate upon aging to 7 months, in contrast to the progressive deterioration of cardiac function and morbidity of a transgenic mouse model with 10-fold overexpression of dog cardiac calsequestrin (24, 32). Upon ablation of phospholamban, both +dP/dt and -dP/dt values of calsequestrin overexpressing hearts were significantly increased to levels even higher than those in the WT littermates. Similar effects were observed in older (7 month) mice, suggesting the functional benefit of phospholamban ablation over the long term. Furthermore, there was no difference in the intrinsic heart rate across the three groups with different ages.


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Fig. 2.   Effects of phospholamban ablation on contractile parameters of isolated hearts overexpressing calsequestrin. A, Langendorff-perfused hearts from 2- and 7-month-old mice demonstrated depressed maximal rate of contraction (+dP/dt) in calsequestrin overexpressing (CSQOE) mice with no changes in the maximal rate of relaxation (-dP/dt) and the intrinsic heart rate (HR), compared with wild-types (WT). Preparations from calsequestrin overexpressing littermates with phospholamban deficiency (CSQOE/PLBKO) indicated significant increases in +dP/dt and -dP/dt without alterations in HR, compared with WT and CSQOE. B, isolated hearts from 4-month-old mice were perfused with increasing concentrations of isoproterenol (Iso). In CSQOE hearts, 30-300 nM Iso elicited pulsus alternans. Therefore, a 10-s average during an apparent peak response was used as a representative value (points in parentheses). Values are mean ± S.E. (n = 3-5). *, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after two-way ANOVA).

Another important question is whether beta -adrenergic agonists can stimulate the contractile parameters of the CSQOE/PLBKO hearts, as phospholamban is postulated to be a major player in beta -adrenergic responses (13-15). Thus, we examined the effects of isoproterenol stimulation in Langendorff-perfused hearts (Fig. 2B). The positive chronotropic effects of isoproterenol were similar among WT, CSQOE, and CSQOE/PLBKO groups. beta -Agonist stimulation also increased the +dP/dt and -dP/dt values in all three groups, including the hyperdynamic CSQOE/PLBKO (33). However, administration of isoproterenol (30-300 nM) was associated with induction of pulsus alternans in CSQOE hearts, presumably due to the positive chronotropic effect (34). The CSQOE/PLBKO littermates did not display such mechanical abnormalities, and the maximally stimulated ±dP/dt parameters as well as their EC50 values for isoproterenol stimulation were similar between WT and CSQOE/PLBKO.

Rescue of Ca2+ Handling Defects in Calsequestrin Overexpressing Myocytes-- Stable Ca2+ tolerant cardiomyocytes could be achieved with voltage clamping and manipulating the intracellular ionic environment (17, 26, 27). Since caffeine-induced SR Ca2+ release fully saturates the fluorescent dye signal in calsequestrin overexpressing myocytes (35), the INCX current was used to estimate the SR Ca2+ content. When INCX was measured in the absence of caffeine by replacing external Na+ with Li+, the average values of outward INCX current densities were 0.99 ± 0.06 pA/pF (n = 31), 0.90 ± 0.06 pA/pF (n = 32), 0.91 ± 0.06 pA/pF (n = 13), in WT, CSQOE, and CSQOE/PLBKO, respectively. This suggests that the functional activity of Na+-Ca2+ exchanger was not significantly altered in CSQOE or CSQOE/PLBKO, compared with WT myocytes. In subsequent studies, caffeine (5 mM) was added to the external solution in the presence of Na+ to examine whether phospholamban ablation increased the SR Ca2+ content in myocytes overexpressing calsequestrin. Rapid application of caffeine elicited inward INCX currents by sarcolemmal Na+-Ca2+ exchange (Fig. 3A). The peak current density of caffeine-induced INCX currents was increased in CSQOE myocytes, indicating high SR Ca2+ storage, as previously described (23). Phospholamban ablation resulted in further increases in the SR Ca2+ storage, as evidenced by the higher caffeine-induced INCX current density of CSQOE/PLBKO, compared with the CSQOE myocytes (Fig. 3B).


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Fig. 3.   Comparison of the Na+-Ca2+ exchanger currents (INCX) activated by a rapid application of caffeine in ventricular myocytes. A, representative whole cell INCX currents evoked by caffeine (5 mM) in ventricular cardiomyocytes. Myocytes were voltage-clamped at a holding potential of -40 mV. B, the peak caffeine-induced INCX currents normalized to the cell capacitance to give current densities (CD). WT, wild-type; CSQOE, calsequestrin-overexpressing, CSQOE/PLBKO, calsequestrin-overexpressing and phospholamban-deficient. Values are expressed as mean ± S.E. (n = 17-19); *, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after one-way ANOVA).

To gain further insights into the mechanisms underlying the rescue effects of phospholamban ablation, whole cell ICa measurements were performed in the three models (Fig. 4A). The current-voltage relations of CSQOE and CSQOE/PLBKO were similar to WT (data not shown). However, the current density of peak L-type Ca2+ current (ICa) at 2 mM external Ca2+ concentration was significantly reduced in CSQOE myocytes, consistent with previous observations in the FVB/N genetic background (23). Phospholamban ablation resulted in significant increases in the current density of peak ICa, although these values were lower than WT (Fig. 4B). Furthermore, the inactivation kinetics of ICa, which was significantly slower in CSQOE myocytes, was restored upon phospholamban ablation (Fig. 4B).


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Fig. 4.   Properties of L-type Ca2+ currents (ICa) in transgenic mouse ventricular myocytes. A, representative whole cell ICa currents recorded in ventricular cardiomyocytes. Currents were elicited from a holding potential of -50 to +10 mV. B, peak current densities (CD) and half-inactivation times (t1/2) of ICa. C, ICa CD versus ICa t1/2 relations in mouse ventricular myocytes obtained using various concentrations of extracellular Ca2+ (1-20 mM). Values are expressed as mean ± S.E. (n = 17-78); *, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after one-way ANOVA).

We have previously shown that ICa inactivation in mouse myocytes correlates with the local increase in Ca2+ released from the SR, which promotes Ca2+-dependent inactivation. Thus, the inactivation kinetics of ICa can be used to evaluate the efficiency of ICa-induced SR Ca2+ release (17, 26). To elucidate the functional coupling of L-type Ca2+ channels and the SR Ca2+ release, the correlations between densities and inactivation time courses of ICa were examined by varying the external Ca2+ concentration (1-20 mM). As shown in Fig. 4C, the relation between the peak current density and the half-inactivation time was shifted upward in CSQOE, reflecting an inability of ICa to trigger SR Ca2+ release. In contrast, CSQOE/PLBKO and WT myocytes exhibited similar relations between the maximal ICa density and the half-inactivation time, suggesting that phospholamban ablation restored the ability of ICa to trigger SR Ca2+ release.

To examine the stimulatory effects of beta -adrenergic agonists on ICa, 5 mM EGTA in the pipette solution was replaced with 10 mM BAPTA, a more rapid Ca2+ chelator. This replacement negates the enhanced ICa inactivation induced by SR Ca2+ release (17, 26, 27). The peak ICa densities in WT, CSQOE, and CSQOE/PLBKO myocytes were 12.5 ± 0.5 pA/pF (n = 51), 6.0 ± 0.3 pA/pF (n = 38), and 11.6 ± 1.1 pA/pF (n = 43), respectively. Potentiation of ICa was then examined in the presence of various concentrations of isoproterenol, and in the absence or presence of isobutylmethylxanthine (IBMX: 100 µM), a phosphodiesterase inhibitor (Fig. 5A). Perfusion with isoproterenol increased the current amplitude in all groups and the EC50 values of peak ICa were similar among them (Fig. 5B). Although the relative increase of current amplitude was more prominent in CSQOE cells, their ICa density was significantly smaller in the presence of isoproterenol (1 µM), compared with WT or CSQOE/PLBKO cells. Additional application of IBMX enhanced isoproterenol-promoted ICa in all groups, although the current density in CSQOE myocytes remained smaller, compared with WT or CSQOE/PLBKO myocytes (Fig. 5C). Therefore, overexpression of murine calsequestrin or subsequent ablation of phospholamban did not lead to low responsiveness to beta -adrenergic stimulation and/or high phosphodiesterase activity.


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Fig. 5.   Effects of isoproterenol (Iso) and isobutylmethylxanthine (IBMX) on ICa in transgenic mouse ventricular myocytes. A, typical L-type Ca2+ currents (ICa) recorded in ventricular cardiomyocytes. The currents were elicited by voltage-clamp steps from a holding potential of -50 to 0 mV. The currents after the application of isoproterenol (Iso: 1 µM) and subsequent addition of IBMX (100 µM) were superimposed. B, concentration-dependent effects of Iso on ICa in WT, CSQOE, and CSQOE/PLBKO myocytes. The peak current amplitude was normalized to myocyte size (pA/pF) and plotted against Iso concentrations. C, summarized data of the effects of Iso (1 µM) plus IBMX (100 µM) on ICa in WT, CSQOE, and CSQOE/PLBKO myocytes. Values are mean ± S.E. (n = 18-51); *, p < 0.05 versus WT (Fisher's LSD after one-way ANOVA).

Rescue of Action Potential Prolongation in Calsequestrin Overexpressing Myocytes-- A major electrophysiological abnormality, observed in a variety of experimental models of myocardial disease, as well as human heart failure, is action potential prolongation. Thus, we examined the characteristics of action potentials in isolated ventricular myocytes from the three groups. WT myocytes displayed a brief action potential with a rapid initial phase of repolarization without a discernible plateau phase (Fig. 6), similar to previous observations in adult mouse ventricular myocytes (27). However, the action potential duration, quantified at 50% repolarization (APD50), was significantly longer in CSQOE (35.7 ± 5.7 ms, n = 5), compared with WT (17.0 ± 1.4 ms, n = 22) myocytes. Interestingly, the CSQOE/PLBKO myocytes displayed APD50 values (19.8 ± 1.7 ms, n = 11) similar to WTs. There was no significant difference in the resting membrane potentials among the three groups (WT, -69.2 ± 0.4 mV, n = 22; CSQOE, -70.2 ± 0.6 mV, n = 5; CSQOE/PLBKO, -70.0 ± 0.6 mV, n = 10).


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Fig. 6.   Effects of phospholamban ablation on action potentials in calsequestrin overexpressing myocytes. Membrane potential was recorded in the current clamp mode with the patch electrode filled with a K+-rich internal solution. The external solution was normal Tyrode solution. Myocytes were stimulated at 0.2 Hz through the patch pipette.

SR-associated Proteins-- Calsequestrin overexpression was previously shown to result in altered expression of several SR Ca2+ cycling proteins (23). Thus, quantitative immunoblotting was utilized to determine whether phospholamban ablation might restore these changes. The protein levels of the SR Ca2+-ATPase were increased in parallel with up-regulation of phospholamban in CSQOE hearts, but ablation of phospholamban prevented the compensatory up-regulation of the SR Ca2+-ATPase (Table I). The ryanodine receptor levels, which were decreased in CSQOE hearts, were further reduced in CSQOE/PLBKO hearts, similar to previous observations in phospholamban-deficient mice (16). Furthermore, the levels of GRP78 (78-kDa glucose-regulated protein, also known as BiP) and calreticulin, which are Ca2+-binding proteins localized in the lumen of the endoplasmic reticulum (ER) or the SR, were up-regulated by 3.4- and 2.0-fold in CSQOE, respectively. These proteins are known to be molecular chaperones and to play a role in the storage of the exchanging pool of ER Ca2+ in cultured cells (36, 37). They are also known to be up-regulated in response to accumulation of mis- or unfolded ER proteins by ER stress, such as perturbation of Ca2+ homeostasis (38, 39). Phospholamban ablation abolished the increases in GRP78 and calreticulin, which may indicate a relative reduction in ER/SR stress in CSQOE/PLBKO versus CSQOE.

                              
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Table I
Relative levels of SR/ER-associated proteins in cardiac ventricles
Protein levels in cardiac homogenates from wild-type (WT), calsequestrin overexpressing (CSQOE), and calsequestrin-overexpressing as well as phospholamban-deficient (CSQOE/PLBKO) mice were determined using quantitative immunoblotting. Values were normalized to the average level of the specific protein in wild-type hearts. Values represent the mean ± S.E. (n = 7-8).

Cardiac Hypertrophy-- One of the characteristics of hypertrophy is the increase in heart muscle mass, associated with reactivation of a fetal gene program, to compensate for increases in workload. To assess whether improvement of cardiac function in CSQOE/PLBKO mice is accompanied by rescue of the hypertrophic response, ventricular and atrial weights were evaluated. In 2-3-month-old CSQOE mice, the wet ventricular/body weight ratio was increased approximately by 35%, compared with WT (Fig. 7), in agreement with previous observations in the FVB/N genetic background (23). Examination of gross cardiac morphology revealed enlargement of the left atrium (data not shown) and the atrial/body weight ratio was increased by 38%. These increases in weight ratios were not progressive at least up to 7 months of age. Ablation of phospholamban restored the atrial/body weight ratio to WT levels, but it did not reverse the increases in the ventricular/body weight ratio. Two-way ANOVA indicated that the wet tissue/body weight ratios of the CSQOE/PLBKO were not dependent on age. Furthermore, these alterations did not reflect changes in body weights, which were similar among the three genotypes with the same age.


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Fig. 7.   Cardiac weights in transgenic mice. Blotted weights of cardiac ventricles and atria from 2-3- and 7-month-old WT, CSQOE, and CSQOE/PLBKO mice were normalized to the respective body weights. There was no difference in body weight across the groups with similar ages. Values represent the mean ± S.E. (n = 13-21 (2-3-month-old) or 4-6 (7 months old)); *, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after two-way ANOVA).

Ventricular myocyte size was also assessed by cell capacitance of patch-clamped isolated cells. CSQOE myocytes were significantly larger than WTs, but phospholamban ablation restored cell capacitance to WT levels (WT, 140 ± 3 pF; CSQOE, 167 ± 4* pF; CSQOE/PLBKO, 142 ± 3# pF, n = 76-107, *, p < 0.05 versus WT, #, p < 0.05 versus CSQOE). Furthermore, examination of cross-sectional areas of ventricular myocytes indicated significant increases upon calsequestrin overexpression and reversal upon phospholamban ablation (WT, 319 ± 27 µm2; CSQOE, 409 ± 20* µm2; CSQOE/PLBKO, 354 ± 12# µm2, n = 6, *p < 0.05 versus WT; #, p < 0.05 versus CSQOE).

The increase in ventricular/body weight ratio of the CSQOE mice was associated with increased expression of a fetal gene program (Fig. 8), including ANF, alpha -skeletal actin, and beta -myosin heavy chain. Phospholamban deficiency reversed the increases in the mRNA levels of ANF and alpha -skeletal actin in the CSQOE/PLBKO but did not influence the expression of beta -myosin heavy chain levels. These findings suggest that ventricular myocyte hypertrophy induced by calsequestrin overexpression is rescued upon phospholamban ablation.


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Fig. 8.   Attenuation of the hypertrophy gene program in calsequestrin overexpressing myocardium by phospholamban ablation. A, mRNA dot-blot analysis demonstrated increased transcript levels of beta -myosin heavy chain (beta -MHC), ANF, and skeletal alpha -actin (SK-actin), but no significant changes in the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in ventricles of calsequestrin overexpressing (CSQOE) mice, compared with wild-type (WT) littermates. Note that the mRNA levels of ANF and SK-actin in calsequestrin overexpressing littermates with phospholamban ablation (CSQOE/PLBKO) were comparable to the WT. B, quantitative grouped analysis of mRNA expression. Values are the mean ± S.E. (n = 3). *, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after one-way ANOVA).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Defects in SR Ca2+ uptake and release are common features of hypertrophied and failing animal and human myocardia, although the mechanisms underlying the etiology of these defects have not been clear. Furthermore, the contribution of impaired SR Ca2+ cycling to the onset and progression of the hypertrophic response is unknown. To address this question, the current study utilized the calsequestrin overexpressing model with cardiac hypertrophy (23) and introduced phospholamban-deficient alleles to improve the SR Ca2+ transport properties. Phospholamban ablation did not alter the levels or the localization of the overexpressed calsequestrin, which was associated with the terminal cisternae at the z-lines, similar to endogenous calsequestrin. However, the contractile parameters were restored to wild-type levels in vivo and they were super-rescued in Langendorff-perfused hearts, upon ablation of phospholamban. The observed differences in the degree of cardiac function enhancement between the intact animal and the isolated organ levels are presumably due to differences in external loading conditions. This functional improvement was at least partially due to enhanced SR Ca2+ storage in phospholamban-deficient hearts, as estimated by the caffeine-induced INCX. Calsequestrin overexpression has been previously shown to increase the SR Ca2+ content but this Ca2+ pool was not accessible for release (22, 23). However, upon phospholamban ablation, the additional Ca2+ load, accompanied by the enhanced SR Ca2+ uptake, is likely to increase the intraluminal free SR Ca2+ concentration, which is associated with higher amounts of Ca2+ released (19). In addition, the density of ICa, which was decreased in calsequestrin overexpressing myocytes, was partially restored in CSQOE/PLBKO cells, leading to higher activation of the Ca2+-induced SR Ca2+ release despite the reduced levels of the ryanodine receptor, elicited by phospholamban ablation (16). Moreover, the inactivation kinetics of ICa, which are regulated by the Ca2+ influx as well as SR Ca2+ release (17, 26) were rescued and the ICa density-inactivation relation of CSQOE/PLBKO was similar to the WTs. These results suggest that phospholamban ablation restored the impaired SR Ca2+ release properties or defective excitation-contraction coupling of calsequestrin overexpressing cardiomyocytes. Furthermore, prolongation of the action potential, observed in CSQOE myocytes, which may reflect alterations in the transient outward K+ currents (Ito) and possible induction of arrhythmias (27), was restored upon phospholamban ablation.

Cardiac-specific overexpression of dog cardiac calsequestrin has been suggested to attenuate beta -adrenergic responses and enhance myocyte phosphodiesterase activities (24, 32). However, our results indicated that responsiveness of ICa to isoproterenol and/or IBMX was not decreased in cardiomyocytes overexpressing murine cardiac calsequestrin (CSQOE), as well as the CSQOE/PLBKOs, suggesting that the smaller basal current density observed in CSQOE myocytes is not directly mediated by alterations in beta -adrenoreceptor signaling pathway or phosphodiesterase activity, but rather by an indirect effect associated with cardiac hypertrophy. The reasons for these apparent discrepancies between the dog and mouse cardiac calsequestrin overexpressing models may be due to differences in their genetic backgrounds (23, 30-32, 40, 41).

The increases in the expression levels of: (a) calreticulin, which has also been reported to increase in pressure overload hypertrophy (42) and may contribute to increased ER/SR Ca2+ buffering; (b) GRP78, which is another Ca2+-binding molecular chaperone in the ER/SR; and (c) SR Ca2+-ATPase, which constituted an important compensatory response for SR function in CSQOE hearts, were reversed by phospholamban deficiency, indicating a reduction in ER stress or restored ER/SR Ca2+ homeostasis in CSQOE/PLBKO hearts. However, further examinations are needed to reveal the mechanisms by which calsequestrin buffers SR luminal Ca2+, and the effects of phospholamban ablation on free luminal Ca2+ and ryanodine receptor gating properties.

In the calsequestrin overexpressing heart as well as a variety of other experimental models, cardiac hypertrophy was associated with increased expression of fetal genes (23, 43-45). Interestingly, the up-regulation of alpha -skeletal actin and ANF mRNA was reversed by phospholamban ablation. The physiological and pathological roles of increases in alpha -skeletal actin transcripts in cardiac hypertrophy are currently unclear. However, the reduction of ANF mRNA levels by phospholamban ablation may reflect the recovery of cardiomyocytes from a pathological state, since ANF is known to be a sensitive indicator of cardiac pathogenesis rather than the degree of hypertrophy (44). The increases in the slow beta -myosin heavy chain transcript, although relatively small (~2-fold), were not normalized and were expected to contribute to attenuation of contraction rates. However, phospholamban ablation and the restored SR function appeared to overcome the effects of increased beta -myosin heavy chain expression, and the cardiac contractile parameters in CSQOE/PLBKO hearts were at least as high as the WTs. Consistent with the reversal of ANF and alpha - skeletal actin increased expression, the cell capacitance and cross-sectional area of myocytes were restored to normal levels upon phospholamban ablation. Remarkably, these alterations did not reflect similar observations for the ventricular/body weight ratio, which remained high in CSQOE/PLBKO mice, suggesting an increase in the number of myocytes in these hearts. Taken together, these findings suggest that ablation of phospholamban or concomitant improvement of SR Ca2+ handling may shift the mode of ventricular myocyte growth from hypertrophy to hyperplasia, possibly by reducing trophic factors for the adaptive response to Ca2+ handling defects or by increasing cellular mitotic factors.

Phospholamban deficiency also resulted in significant reduction of atrial mass in the calsequestrin overexpressing mice. The reduction of atrial mass suggested that stress on the atria was attenuated, presumably due to the enhanced ventricular function. Enlargement of left atrial size in a hypertrophic heart is postulated as a predictor of atrial fibrillation and thromboembolism leading to stroke, which is associated with increased risk of cardiovascular mortality (46-49). Therefore, inhibition of phospholamban function may not only improve ventricular performance but also prevent atrial-induced morbidity.

Recently, phospholamban ablation was shown to rescue the depressed function and heart failure phenotype in an MLP-deficient mouse model, which displays many phenotypic features of human dilated cardiomyopathy (50). Cardiac-specific overexpression of the beta -adrenergic receptor kinase inhibitor was also reported to improve cardiac function in the same model of cardiomyopathy (51). Since phospholamban is the major substrate in the cardiac beta -adrenergic pathway, it is interesting to propose that at least a part of the beneficial effects by the latter genetic manipulation may be mediated by the phosphorylation level of phospholamban. The rescue effects of phospholamban ablation were maintained over the long-term without increasing the intrinsic heart rate in both the calsequestrin overexpressing and MLP-deficient models (50). However, ablation of phospholamban may not be beneficial for all hypertrophic phenotypes, especially when impaired Ca2+ cycling is not associated with the end point (52, 53). Furthermore, it remains to be determined whether inhibition of phospholamban activity in a tissue-specific and inducible manner may still rescue cardiac function and remodeling, when applied subsequent to the onset of hypertrophy and failure.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL26057, HL52318, P40RR12358, and HL64018 (to E. G. K.), GM54169 and HL61476 (to A. Y.), the American Heart Association, Ohio Valley Affiliate (to A. Y.), and a Ministry of Health and Welfare (Japan) grant (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Contributed equally to the results of this work.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575. Tel.: 513-558-2377; Fax: 513-558-2269; E-mail: kraniaeg@email.uc.edu.

Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M006889200

    ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; CSQOE, calsequestrin overexpressing; CSQOE/PLBKO, calsequestrin overexpressing in phospholamban knockout background; WT, wild-type; INCX, Na+-Ca2+ exchanger current; ICa, L-type Ca2+ current; ANF, atrial natriuretic peptide; ER, endoplasmic reticulum; BAPTA, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; IBMX, isobutylmethylxanthine; LSD, least significant difference.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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