INTRODUCTION

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THE PLASMA MEMBRANE CALMODULIN-dependent calcium ATPase (PMCA) is a ubiquitous Ca²⁺-transporting enzyme extruding Ca²⁺ from the cell (24). In nonexcitable cell types, PMCA is the only known mechanism for Ca²⁺ extrusion (4). In excitable cells, which express the high capacity Na⁺/Ca²⁺ exchanger, the activity of PMCA in vitro is rather low compared with Na⁺/Ca²⁺ exchanger (2). PMCA isoforms 1, 2, and 4 are expressed in the myocardium (4, 16), and it has been suggested that PMCA, due to its high affinity on Ca²⁺, may play a role in fine tuning Ca²⁺ in the final phase of diastole in the heart (3). In transgenic (TG) rats, overexpression of PMCA does not induce major changes in baseline cardiac contractility or contractile parameters of peak performance obtained by volume loading, and it has also been reported that PMCA is not involved in beat-to-beat regulation of myocardial function (15). On the other hand, PMCA has been suggested to have a role in growth and differentiation processes in myoblasts as well as in other cell types in vitro (14). Altered growth and differentiation responses to phenylephrine and isoproterenol have been documented in PMCA overexpressing neonatal cardiac myocytes in vitro (15). PMCA has been shown to localize in caveolae, small 50- to 100-nm plasma membrane invaginations, containing receptors for endothelin-1 (ET-1) and various other ligands (6, 15, 36). However, the physiological function of PMCA and its role in the cardiac responses to hypertrophic signals in myocardial cells in vivo are not known.

In the present study, to characterize the role of PMCA in the hypertrophic response, we measured left ventricular c-fos and B-type natriuretic peptide (BNP) mRNA levels after treating TG rat hearts overexpressing PMCA with ET-1. ET-1 induces hypertrophic responses both in vivo and in vitro (7, 20), and in isolated perfused rat heart preparation, ET-1 activates the gene expression of c-fos and BNP within 2 h (26). We also evaluated the induction of adrenomedullin (AM) gene expression in response to ET-1 infusion in non-TG (NTG) and PMCA-overexpressing rat hearts. AM may function as an autocrine and/or paracrine factor in the heart (38), and both hemodynamic overload (31) and ET-1 (26) have been reported to increase rapidly AM mRNA levels in left ventricles of normal rat hearts. Finally, as another stimulus for early induction of cardiac gene expression, we loaded hearts overexpressing PMCA by increased coronary perfusion pressure, which will distend the left ventricular wall and induce a hypertrophic gene expression response (12, 26).

	MATERIALS AND METHODS

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Experimental animals. The TG rat line, designated as PMCA rats, was carrying the human PMCA isoform 4CI (also called PMCA 4b) cDNA under control of the ventricle-specific rat myosin light chain-2 promoter (15). The total amount of PMCA protein in the adult ventricles was 1.6-fold compared with control animals. Furthermore, a 1.8-fold increase in the Ca²⁺-ATPase activity in rats overexpressing PMCA was noted (15). The NTG control rats from the Sprague-Dawley-Hannover strain were from Møllegaard Experimental Animal Center (Skensved, Denmark). Body weights of 2-mo-old male rats were 405 ± 7 (n = 18) and 398 ± 5 g (n = 23) for NTG and PMCA TG rats, respectively. The experimental design was approved by the Animal Use and Care Committee of the University of Oulu.

Isolated perfused heart preparation. The isolated perfused rat heart preparation was similar to that described previously (32). Briefly, 20 min after intraperitoneal injection of heparin (500 IU/kg body wt), rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique. The hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O₂-5% CO₂ at 37°C. The composition of the buffer was (in mmol/l) 113.8 NaCl, 22.0 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.1 MgSO₄, 2.5 CaCl₂, and 11.0 glucose. The hearts were stimulated via the pulmonary artery cannula and the cannula in the inferior vena cava (11 V, 0.5 ms) using a Grass stimulator to increase the heart rate to 300 beats/min. During the equilibration period (50 min) the hearts were perfused at a constant flow rate of 5 ml/min. This flow rate results in maximal vasodilatation and allows detection of the vasoconstrictor effects of infused substances (38). After a 10-min control period, either ET-1 (1 nmol/l; Phoenix) or vehicle infusion was begun at a rate of 0.5 ml/min. In pressure-overloaded hearts, the coronary flow rate was increased to a level of 20 ml/min. Previously, this isolated perfused heart preparation has been found to be a sensitive method for studying inotropic action of various agents (23) as well as cardiac gene expression responses to different stimuli (26). Contractile force (apicobasal displacement) was obtained by connecting a force-displacement transducer (Grass Instruments, model FT03) to the apex of the heart at an initial preload stretch of 2 g as previously described (38). Perfusion pressure was measured by a pressure transducer (Micron Instruments, model MP-15) situated on a side arm of the aortic cannula. All recordings were made with the use of a Grass 7DA polygraph. At the end of each experiment, the hearts were weighed and both ventricles and atria were immersed in liquid nitrogen and stored at 70°C until assayed.

Isolation and analysis of cytoplasmic RNA. RNA was isolated from atria and ventricles by the guanidine thiocyanate-CsCl method (5). For the RNA Northern blot and dot blot analyses, 20-µg samples of the RNA were transferred to Amersham Hybond N+ nylon membranes. A 390-bp fragment of rat BNP cDNA probe (30) (a generous gift from Dr. K. Nakao, Kyoto University School of Medicine, Kyoto, Japan), full-length rat atrial natriuretic peptide (ANP) cDNA probe (10) (a generous gift from Dr. P. L. Davies, Queen's University, Kingston, Ontario, Canada), PCR amplified rat AM cDNA probe (nucleotides 287-736) (31), full-length cDNA probe complementary to GAPDH (11), cDNA probe made by RT-PCR for rat c-fos (nucleotides 231-1280), and cDNA probe complementary to rat 18S ribosomal RNA (29) were labeled with [³²P]dCTP with T7 Quick Prime Kit (Pharmacia LKB Biotechnology), and the membranes were hybridized and washed as described previously (27). Membranes were exposed to Phosphor screens and scanned with Phosphor Imager (Molecular Dynamics).

Radioimmunoassays of BNP and AM. The AM and BNP radioimmunoassays were performed as previously described (27, 31). The sensitivities of the AM and BNP assays were 1 and 2 fmol/tube, respectively. The intra- and interassay variations were <10 and 15%, respectively. Serial dilutions of tissue and plasma extracts showed parallelism with the standards. Tissue AM and BNP are expressed as a concentration per milligram wet weight.

Statistics. The results are expressed as means ± SE. Student's t-test was used for the comparison between two groups. The hemodynamic variables and immunoreactive (ir) peptide levels were analyzed with one-way ANOVA followed by Student-Newman-Keuls post hoc test. Repeated-measures ANOVA was used for multivariate analysis. Differences at the 95% level were considered statistically significant.

	RESULTS

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Hemodynamic parameters in isolated perfused PMCA rat hearts. In a previous study, using in vivo hemodynamic measurements, there were no differences between PMCA-overexpressing rats and NTG littermates in cardiac function (15). In the isolated perfused rat heart model used here, baseline hemodynamic parameters were similar between NTG and PMCA rat hearts (Table 1). Administration of ET-1 resulted in an equal 120 ± 12 and 123 ± 23 mmHg increase in coronary perfusion pressure in NTG and PMCA rat hearts, respectively (Fig. 1). ET-1 was previously reported to have an inotropic effect on the heart (19). In the present study, there was a 24 ± 6 and 37 ± 7% increase (P < 0.05 for both vs. baseline values) in contractile force after 20 min of ET-1 infusion in NTG and PMCA rat hearts, respectively (Fig. 1). There was no significant difference in contractile force between NTG and PMCA rats in response to ET-1 infusion. When ET-1 infusion was continued, the contractile force gradually decreased, as reported previously (1). The attenuation of the inotropic response was most likely due to direct action of ET-1 on cardiac myocytes, because the flow rate was kept constant. Left ventricles weighed 532 ± 15 and 539 ± 15 mg in NTG and PMCA animals, respectively. The respective ventricular-to-body weight ratios were 1.32 ± 0.03 and 1.34 ± 0.03 mg/g, showing that 2-mo-old PMCA rats did not have left ventricular hypertrophy.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Plots showing changes in contractile force (A) and perfusion pressure (B) in nontransgenic (NTG) and plasma membrane calmodulin-dependent calcium ATPase (PMCA) rats in response to endothelin (ET)-1 (1 nmol/l) or increased flow rate (load). Solid line, NTG rats; dashed line, PMCA rats. , NTG control; , NTG ET-1; , NTG load; , PMCA control; , PMCA ET-1; , PMCA load. There was no significant difference between NTG and PMCA rats in response to either of the treatments. Both ET-1 and increased flow rate produced a significant increase in contractile force at the 20 min time point in both NTG and PMCA rat hearts (P < 0.05; 1-way ANOVA followed by Student-Newman-Keuls post hoc test)(A). As seen in B, perfusion pressure increased significantly with both ET-1 and increased flow rate (P < 0.001; repeated-measures ANOVA).

BNP response to ET-1 in PMCA rat hearts. To evaluate the possible role of PMCA in regulation of cardiac gene expression, both PMCA-overexpressing and NTG rat hearts were perfused for 2 h with 1 nmol/l ET-1. In NTG rats, BNP mRNA levels in left ventricles increased 2.0-fold in response to ET-1 infusion (P < 0.01) (Figs. 2 and 3). In contrast, in PMCA-overexpressing rat hearts, ET-1 did not induce any significant increase in left ventricular BNP mRNA levels (Figs. 2 and 3). Accordingly, release of ir-BNP into the perfusate in response to ET-1 infusion remained unchanged in PMCA transgenic rats, whereas a 1.5-fold increase in BNP secretion (P < 0.05) compared with baseline levels in NTG rats was noted (Fig. 4). Baseline concentration of ir-BNP in the perfusate was 1.7 ± 0.2 and 1.6 ± 0.2 fmol/ml in control and TG rats hearts, respectively. Also, baseline BNP mRNA levels were similar in left ventricles of NTG and PMCA animals (0.79 ± 0.08 densitometric units, n = 4 vs. 0.72 ± 0.06 densitometric units, n = 7, respectively). Furthermore, left ventricular ir-BNP levels in control and TG rat hearts were comparable, and ET-1 infusion for 2 h did not induce significant changes in tissue ir-BNP levels of either strain (data not shown). This latter finding is consistent with our previous results (26) and may be explained by rapid secretion of BNP from the ventricles promptly after its synthesis (30). As expected (26), ANP mRNA levels remained unaltered in both TG and NTG rat hearts in response to 2-h stimulation with ET-1 (Fig. 2).

View larger version (97K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis showing the effects of ET-1 (1 nmol/l) and increased coronary flow (Load) on left ventricular adrenomedullin (AM), atrial natriuretic peptide (ANP), c-fos, B-type natriuretic peptide (BNP) and GAPDH mRNA levels of NTG (A) and PMCA (B)-overexpressing transgenic rat hearts in isolated perfused hearts.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Bar graphs showing the effects of ET-1 (1 nmol/l) and increased coronary flow for 2 h on left ventricular BNP mRNA in NTG (A) and PMCA (B)-overexpressing transgenic rats in isolated perfused hearts. mRNA results are expressed as a ratio of BNP to GAPDH as determined by Northern blot analysis. Data are means ± SE. For number of experiments in each group, see Table 1. *P < 0.05, P < 0.01, and P < 0.001 vs. control (Student's t-test).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of ET-1 (1 nmol/l) for 2 h on immunoreactive (ir)-BNP (A) and ir-AM (B) levels in perfusate of NTG and PMCA-overexpressing transgenic rats in isolated perfused hearts. Results are expressed relative to baseline secretion rate during control period. Solid line, NTG rats; dashed line, PMCA rats. , NTG control; , NTG ET-1; , PMCA control; , PMCA ET-1. Data are means ± SE. For number of experiments in each group, see Table 1. *P < 0.05 vs. NTG control and vs. PMCA ET-1; P < .005 vs. SD control (repeated-measures ANOVA).

Effect of ET-1 on AM and c-fos gene expression in PMCA rat hearts. To evaluate whether the impaired response to ET-1 in PMCA-overexpressing rat hearts is specific to BNP gene, we studied the effects of ET-1 on left ventricular AM and c-fos mRNA levels. A 2-h perfusion with ET-1 resulted in 1.7- and 2.5-fold increase in AM and c-fos mRNA levels in left ventricles of NTG rat hearts, respectively (Figs. 2 and 5). This early induction of AM and c-fos gene expression was almost completely abolished in left ventricles of PMCA rats (Fig. 5). Baseline concentration of ir-AM in the perfusate was 0.10 ± 0.02 and 0.13 ± 0.03 fmol/ml in NTG and PMCA animals, respectively. ET-1 induced a 2.0-fold increase in secretion of ir-AM in NTG animals but not in PMCA rat hearts (P < 0.05, ET-1-treated NTG vs. PMCA rats) (Fig. 4). Left ventricular ir-AM concentrations were 246 ± 6 and 266 ± 8 fmol/g in NTG and PMCA hearts, respectively (P = NS).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of ET-1 (1 nmol/l) for 2 h on left ventricular AM (A) and c-fos (B) mRNA in NTG and PMCA-overexpressing transgenic rats in isolated perfused hearts. Results are expressed as a ratio of AM or c-fos mRNA to GAPDH as determined by Northern blot analysis. Data are means ± SE. For number of experiments in each group, see Table 1. *P < 0.05 vs. control (Student's t-test).

Responses to ET-1 in atria of PMCA-overexpressing rat hearts. Next, we examined whether the induction of BNP gene expression in response to ET-1 in atria, where no PMCA overexpression is present (data not shown), is similar in NTG and PMCA rats. In the left atria, perfusion with 1 nmol/l ET-1 resulted in 1.6- and 1.7-fold increases in BNP mRNA levels in NTG and PMCA rat hearts, respectively (P < 0.005 vs. vehicle) (Fig. 6). The left atrial concentration of ir-BNP did not differ between NTG and PMCA rats, and ET-1 infusion for 2 h did not induce significant changes in ir-BNP levels of atrial tissue in either strain (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effects of ET-1 (1 nmol/l) for 2 h on left atrial BNP mRNA in NTG (A) and PMCA (B)-overexpressing transgenic rats in isolated perfused hearts. Results are expressed as a ratio of BNP mRNA to 18S as determined by Northern blot analysis. Data are means ± SE. For number of experiments in each group, see Table 1. *P < 0.005 vs. control (Student's t-test).

BNP response to increased perfusion pressure in PMCA-overexpressing rat hearts. Finally, to examine whether the attenuated hypertrophic responses are selective to ET-1, we loaded NTG and PMCA rat hearts by increasing coronary flow from 5 to 20 ml/min (26). This increase in flow resulted in a 120-mmHg increase in coronary perfusion pressure in both PMCA and NTG rat hearts (Fig. 1). The contractility initially increased due to the wall stretch and then decreased gradually after 1 h perfusion (Fig. 1). Left ventricular BNP mRNA levels showed similar 1.6- and 1.5-fold increases (P < 0.01) in response to increased coronary pressure in both strains (Figs. 2 and 3). Concentration of ir-BNP in left ventricles did not change significantly in either group by increasing coronary flow, and in atrial tissue no changes were seen in either BNP peptide or mRNA levels (data not shown).

	DISCUSSION

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The physiological function of the sarcolemmal calcium pump in the myocardium is unknown. Previously, Hammes et al. (15) showed that there are no differences between control and PMCA rats in baseline or volume load increased cardiac performances in vivo. In the present study, baseline contractility was similar in NTG and PMCA hearts in isolated perfused heart preparation. We also did not see any difference in baseline cardiac gene expression, secretion of BNP and AM into the perfusate, and left ventricular-to-body weight ratios between PMCA and control rats. These results support the hypothesis that PMCA-overexpressing animals have no cardiac insufficiency. Hammes et al. (15) also found no differences in patch clamp voltage dependence, activation/inactivation behavior of the L-type Ca²⁺ current, or fast inward Ca²⁺ transients in TG and control cardiomyocytes.

A key finding of the present study was that myocardial overexpression of the PMCA attenuated the hypertrophic response to ET-1 as shown by almost complete abolishment of the early induction of BNP, AM, and c-fos gene expression in left ventricles as well as BNP and AM secretory responses to ET-1. The genetic construct used to generate the TG rat line is expressed under myosin light chain-2 promoter restricting the expression of PMCA to ventricular myocardium (15). As shown in Fig. 6, ET-1-induced early activation of BNP mRNA synthesis remained intact in atrial tissue of TG animals. This further suggests that the alterations seen in ventricular gene expression as well as AM and BNP secretion are due to an increased amount of PMCA present in the ventricles. The vasoconstricting action of ET-1 was not altered in PMCA rats, showing that smooth muscle cells in vascular walls of both lines responded similarly to ET-1.

Previously PMCA was reported to be involved in regulation of growth and differentiation processes in various cell types. In vascular smooth muscle cells and also in ovary cells, the effects of PMCA overexpression were growth and proliferation inhibition (13, 18), whereas in myoblasts, PMCA overexpression resulted in acceleration of differentiation process (14). Hammes et al. (15) studied the response of cultured neonatal cardiomyocytes of this same PMCA-overexpressing rat line to fetal calf serum, phenylephrine, and isoproterenol. They found an increased protein synthesis rate in response to all of these different stimuli in PMCA-overexpressing cells. Meanwhile, in the present study, myocardial overexpression of PMCA attenuated the early induction of cardiac gene expression induced by ET-1 in perfused rat hearts. The reason for this discrepancy remains to be studied, but one possible explanation for the different effects of hypertrophic stimuli on cardiac protein synthesis in cultured cells compared with intact hearts may relate to the experimental conditions of cardiac myocytes under cell culture. For example, the data obtained in isolated neonatal and adult cardiac myocytes suggest that ANG II is a potent stimulus for cardiac c-fos expression (9), although it failed to stimulate this protooncogene in the intact ex vivo perfused adult rat heart (34).

To study further the effects of PMCA overexpression to hypertrophic responses in intact adult heart, we compared the responses in cardiac gene expression stimulated by ET-1 to that produced by mechanical load in an isolated, perfused heart preparation. The response to mechanical stimulus, which here was increased coronary flow, was similar to control hearts, showing that there is no common alteration in myocardial gene expression in PMCA rats in response to hypertrophic stimuli. In view of unchanged baseline mRNA concentrations and peptide secretion, this suggests that there is no general alteration in synthesis and secretion of BNP and AM in PMCA-overexpressing rat hearts. Furthermore, these results also implicate that the signal transduction mechanisms for ET-1-induced hypertrophic responses differ from those activated by ventricular stretch. In agreement with this hypothesis, induction of BNP gene in ventricles in response to mechanical stimulus has been shown to be independent of ET-1 and ANG II (28), and stretch has been shown to increase the release of other growth factors in addition to ET-1 and ANG II, such as transforming growth factor- (25).

It was previously shown that overexpression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2; ~20%) in TG mice results in faster decline of calcium transients and increased cardiac contractility (17). SERCA2 plays a dominant role in the lowering of cytoplasmic calcium levels during cardiac relaxation, whereas PMCA has only a minor role extruding calcium from cytosol after each beat (2). In view of this, it was expected that there is no difference between NTG and PMCA hearts in baseline contractility. In our study, the overexpression of PMCA also had no effect on inotropic responses to ET-1, which suggests that the intracellular signaling mechanisms leading to hypertrophic response and to increased contractility may be different. The hypertrophic effect of ET-1 is known to be mediated by ET_A receptors (33). Activation of ET_A-receptors result in formation of inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol, which induce release of Ca²⁺ and activation of protein kinase C (PKC), respectively, and lead to activation of mitogen-activated protein kinase (MAPK) and other signaling pathways (33, 37). Because ET_A receptors are known to be located in caveolae (6), attenuation of ET-1 response may be due to alterations in caveolar signal transduction (36) induced by PMCA overexpression. Several proteins involved in signal transduction have been localized to caveolae, including Gs, ras, PKC-, MAPK, src tyrosine kinase, and channels such as IP₃-sensitive Ca²⁺ channel (8, 35, 36). The mechanism by which PMCA overexpression could affect caveolar signaling events may involve modification of subcellular Ca²⁺ pools or direct interaction with other caveolar molecules, such as ET_A receptors or other molecules involved in ET-1 signaling process. However, due to the small size of caveolae (50-100 nm), no direct measurements of calcium transients are technically possible at present.

It has been reported that in liver, PMCA is regulated by Gs (21), which can be coupled to ET_A or ET_B receptors (21, 39). It has also been shown that calcium signal provoked by ETs in liver cells is not only due to activation of phospholipase C but also to inhibition of the PMCA, PMCA being coupled to ET_A receptor by Gs (22). In view of this, the attenuated ET-1 response in PMCA-overexpressing rat hearts could be explained simply by the higher amount of PMCA present, resulting in increased capacity to extrude Ca²⁺ from the specific subcellular pool. This hypothesis supports a major role for PMCA in regulating the hypertrophic ET-1 response in myocardium.

In conclusion, the results show for the first time that PMCA plays a physiological role in regulation of myocardial function. Myocardial overexpression of PMCA attenuated early induction of left ventricular BNP, c-fos, and AM gene expression induced by ET-1 but not by increased load. ET-1 infusion for 2 h increased BNP mRNA levels twofold in left ventricles of NTG rats, whereas no increase was noted in PMCA rat hearts. Also, ir-BNP and ir-AM secretory responses to ET-1 were abolished by PMCA overexpression.

Perspectives