INTRODUCTION

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THERE IS ACCUMULATING EVIDENCE that stimulation of Ca²⁺ uptake into the sarcoplasmic reticulum (SR), either by overexpression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a or by downregulation of the inhibitory protein phospholamban, is a promising strategy for support of the failing heart. Transgenic mice overexpressing SERCA2a were more resistant to induction of heart failure by aortic banding or diabetes than wild-type mice (15, 31). Similarly, phospholamban knockout (Plb-KO) mice have no increased mortality and can rescue various transgenic heart failure phenotypes (11, 20, 21, 29). Transfection of myocytes from the failing human heart with adenoviral vectors for either SERCA2a or antisense message for phospholamban enhances contractile function, reversing the slowing of relaxation and depression of the frequency-response curve that are hallmarks of the failing heart (6, 8). Similarly, rescue of contractile function was seen in rats approaching senescence or with heart failure secondary to aortic banding (22, 26). Expected problems related to SR overload resulting from these interventions have not materialized. Instead, the arrhythmogenic potential of increased extracellular Ca²⁺ was reduced after SERCA2a overexpression in rabbit myocytes and cell viability was increased (4). In vivo transfections showed reduced mortality in the rat model of heart failure, with a decrease in incidence of arrhythmias and improved maintenance of ATP levels in the ventricular myocardium (10).

Stimulation of SR Ca²⁺ uptake resembles to some extent the effect of -adrenoceptor agonists, which increase SERCA2a activity by a PKA-induced phosphorylation of phospholamban. However, -adrenoceptor-dependent phosphorylation also affects the L-type Ca²⁺ channel, troponin I, and the SR Ca²⁺ release channel to produce a coordinated increase in force of contraction and acceleration of relaxation (17, 32). It is important to be able to predict the interaction between stimulation of SERCA2a activity by gene transfer and stimulation through the -adrenoceptor cascade, particularly because of the high sympathetic drive in heart failure subjects. From first principles it might be expected that complete reduction in phospholamban levels would prevent the stimulatory effect of -adrenoceptor activation on SR Ca²⁺ uptake. However, reduction in the sensitivity of the myofilaments to Ca²⁺ is also thought to contribute to the acceleration of relaxation by PKA, so that some lusitropic effect of isoproterenol might remain (16). The effect of partial reduction in phospholamban levels is more difficult to predict. Similarly, overexpression of SERCA2a could potentially have a more pronounced effect than relief of phospholamban inhibition, but it is less easy to forecast whether the remaining function would be significantly affected by -adrenoceptor stimulation. The aim of the present study was to compare explicitly the magnitude of the effects of phospholamban downregulation with those of SERCA2a overexpression and the interaction of each of these interventions with -adrenoceptor stimulation. Importantly, this was done in rabbit myocytes, in which the contribution of SERCA2a activity to control of Ca²⁺ movements resembles that in the human heart more closely than does rat or mouse myocardium (2).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Adenoviral vectors. The overall strategy developed by He et al. (14), which involves three steps, was used. First, the SERCA2a or full-length phospholamban antisense sequence cDNA was cloned into the shuttle vector pAdTrack-CMV. Second, the resultant construct was cleaved with the restriction endonuclease PmeI to linearize it. This was then cotransformed with a supercoiled backbone adenoviral vector pAdEasy-1 into Escherichia coli strain BJ5183. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. Third, the recombinant adenoviral construct was cleaved with PacI to expose its inverted terminal repeats and transfected into the packaging cell line, HEK 293. The process of viral production was followed in the packaging cells by visualization of the green fluorescent protein (GFP) reporter that is incorporated into the viral backbone under control of a second cytomegalovirus (CMV) promoter. After 7-10 days, the virus was harvested and then amplified by infecting increasing numbers of packaging cells each time, with a final round using a total of 50 25-cm² flasks. After 2-3 days, the resultant viruses (Ad.SERCA2a.GFP and Ad.PlbAs.GFP) were purified by CsCl banding. A reporter virus producing GFP alone under the control of a CMV promoter was also generated (Ad.GFP). The use of Ad.SERCA2a.GFP, Ad.GFP (4, 8), and Ad.PlbAs.GFP (6) has been described previously.

Isolation, culture, and infection of adult rabbit ventricular myocytes. Hearts were removed from adult male New Zealand White rabbits and retrogradely perfused with a low-calcium (LC) solution [in mM: 120 NaCl, 5.4 KCl, 5 MgSO₄, 5 pyruvate, 20 glucose, 20 taurine, 10 HEPES, and 5 nitrilotriacetic acid (NTA), pH 6.95, preoxygenated with 100% O₂] for 5 min. Type XXIV protease (2 IU/ml; Sigma, Poole, UK), 0.3 mg/ml hyaluronidase (Sigma), and 0.5 mg/ml collagenase (Worthington, Lorne Laboratories, Twyford, UK) were suspended in the LC solution without NTA and with 200 µM Ca²⁺ (pH 7.4). Protease was recirculated through the heart for 2 min, followed by collagenase plus hyaluronidase for 10 min. The left ventricle was then chopped and shaken under 100% O₂ in collagenase plus hyaluronidase for a further 2 × 10 min. The myocytes were washed three times by gentle spinning in Dulbecco's solution (Sigma) containing 10,000 U/ml penicillin-10 mg/ml streptomycin solution (Pen-Strep, GIBCO) and plated in a 12-well culture dish in M199 solution with the addition of 0.2% (wt/vol) bovine serum albumin, 100 mM ascorbate, 5 mM creatine, 5 mM taurine, 2 mM carnitine, 0.1 µM insulin, and Pen-Strep (4). To 10,000 rod-shaped myocytes in each well of a 12-well plate was added 2-10 × 10⁷ plaque-forming units (pfu) of Ad.SERCA2a.GFP, 1-3 × 10⁷ pfu of Ad.PlbAs.GFP, or 2 × 10⁷ pfu of Ad.GFP, and the cells were cultured for 48 h. In each case >90% of myocytes displayed production of GFP.

Functional characterization. Myocytes were superfused with Krebs-Henseleit solution (in mM: 119 NaCl, 1 CaCl₂, 4.7 KCl, 0.94 MgSO₄, 1.2 KH₂PO₄, 2 NaHCO₃, and 11.5 glucose) gassed with 95% O₂-5% CO₂ to pH 7.4. Experiments were carried out at 37°C with field stimulation at 0.5 Hz, and contraction was monitored by a video edge detection device with spatial resolution of 1 in 256 or 512 and a time resolution of 10 or 20 ms. Contraction amplitude, time to peak contraction, and times to 50% and 90% relaxation (RT₅₀ and RT₉₀, respectively) in basal (2 mM) Ca²⁺ were obtained from 5-10 myocytes in each preparation. An individual myocyte was then selected, and a concentration-response curve to isoproterenol was constructed with half-log unit increments of l-isoproterenol-HCl (Sigma) from 0.1 nM until either a maximum had been reached (no increase in contraction amplitude between increments) or arrhythmias were observed. Thapsigargin (Sigma; dissolved in DMSO at a stock solution of 1 mM) was added to contracting myocytes at a final concentration of 1 µM and allowed to act for 10 min. This protocol was sufficient to prevent any uptake or release of Ca²⁺ from the SR (30). Concentration-response curves to isoproterenol were then constructed in the continuing presence of thapsigargin.

Western blotting. For Western blotting, myocytes were cultured by attachment to laminin-coated 35-mm² dishes and nonviable cells were removed by washing 1 h after attachment and immediately before harvest. After 48-h culture with or without viral infection, the myocytes were scraped from the dishes, spun at 500 g for 1 min, and resuspended in PBS. Myocytes were lysed in 15% SDS, 100 mM Tris · HCl (pH 6.8), 40 mM PMSF, and 10 mM EDTA, with vortexing every 5 min for 25 min plus trituration through a fine needle. After centrifugation at 10,000 g, the supernatant was collected and protein concentrations were determined with the Bradford reagent (Bio-Rad protein microassay kit), followed by standard Western blotting techniques. Briefly, lysates were diluted in Laemmli sample buffer (15% SDS) and electrophoresed on acrylamide gels, followed by blotting onto a nitrocellulose membrane (Hybond C; Amersham). Membranes were then exposed to primary mouse monoclonal antibodies for SERCA2a and phospholamban and a secondary anti-mouse Ig peroxidase-linked, species-specific F(ab')₂ fragment from sheep (Affinity Bioreagents, Golden, CO). Binding of secondary antibodies was detected with the ECL system (Amersham).

Statistical analysis. Results for a number of myocytes for a given preparation were pooled, so that n values for statistics refer to preparations except where indicated. Results are expressed as means ± SE or geometric means ± 95% confidence intervals. Comparisons were performed with paired Student t-tests, where appropriate, or group t-tests or one-way ANOVA followed by Fisher's test for pairwise comparison of means.

	RESULTS

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Downregulation of phospholamban: effects on contraction amplitude. Infection of adult rabbit myocytes with adenovirus expressing the antisense sequence for phospholamban (Ad.PlbAs.GFP) was able to decrease phospholamban levels by >50% in 48 h (Fig. 1). This compares with a fivefold overexpression of SERCA2a with the Ad.SERCA2a.GFP virus (4, 8). Ad.PlbAs.GFP did not alter SERCA2a levels (Fig. 1). Changes in contraction amplitude were frequency dependent (Fig. 2). At 0.1 Hz, there was no significant difference between groups, whereas at 2 Hz amplitudes were significantly higher than control (or Ad.GFP infected) for both Ad.PlbAs.GFP- and Ad.SERCA2a.GFP-treated myocytes. Phospholamban downregulation and SERCA2a overexpression were equally effective in increasing contraction amplitude. At 0.5 Hz, the stimulation frequency chosen for the subsequent experiments, there was a small difference in basal contraction that just escaped statistical significance (ANOVA, P = 0.08).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Phospholamban (Plb) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a levels in myocyte preparations treated with Ad.PlbAs.GFP. A: Western blots of SERCA2a (~110 kDa) and monomeric phospholamban (~5 kDa) in uninfected (Con) and infected (As) myocytes. Adjacent Con and As pairs are from the same animal. B: average levels of phospholamban protein in uninfected and Ad.PlbAs.GFP-treated myocyte preparations (arbitrary densitometric units).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Contraction amplitude at increasing stimulation frequency in cultured (48 h) uninfected myocytes (Con; n = 14) and myocytes infected with Ad.GFP (GFP; n = 16), Ad.PlbAs.GFP (Plb-as; n = 17), or Ad.SERCA2a.GFP (SERCA2a; n = 17) for 48 h. Amplitude was significantly different by ANOVA for 2 Hz (P < 0.001) but not 0.1 or 0.5 Hz. * Significantly different from Con or GFP (P < 0.001, Fisher's test).

-Adrenoceptor stimulation and contraction amplitude and arrhythmias. Concentration-response curves to isoproterenol were constructed in control and Ad.PlbAs.GFP- or Ad.SERCA2a.GFP-infected myocytes at 48 h (Fig. 3). There was little difference between groups either in sensitivity of isoproterenol or in the maximum contraction reached. Subtraction of basal contraction did not substantially change the relation between groups (Fig. 3B). For control curves, 70.5% of the concentration-response curves were terminated by arrhythmias at concentrations of 1 µM: the corresponding values for Ad.PlbAs.GFP- or Ad.SERCA2a.GFP-infected myocytes were 60% and 50%, respectively [not significant (NS), ²-test]. For those that did develop arrhythmias below 1 µM, average arrhythmic concentrations did not differ between groups [control: 254 nM (165-640 nM) (geometric mean ± 95% confidence interval), PlbAs: 344 nM (254-797 nM), SERCA2a: 212 nM (58-901 nM); NS].

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves to isoproterenol in adult rabbit myocytes expressed as contraction amplitude (% shortening; A) or % maximum change over basal level (B). , Control, uninfected myocytes (n = 17); , Ad.PlbAs.GFP-infected myocytes (n = 20); , Ad.SERCA2a.GFP-infected myocytes (n = 12). Ad.GFP infection had no significant effect on the isoproterenol concentration-response curve (data not shown).

-Adrenoceptor stimulation and relaxation time. Figure 4 shows RT₅₀ in nine preparations in which responses in Ad.PlbAs.GFP- and Ad.SERCA2a.GFP-treated myocytes were directly compared. A maximally stimulating concentration of isoproterenol (1 µM) was used in these experiments. SERCA2a overexpression was slightly more effective in reducing RT₅₀ than phospholamban downregulation, but the difference was not statistically significant. The effect of isoproterenol alone was similar to that of SERCA2a overexpression. Isoproterenol further significantly reduced RT₅₀ in either Ad.PlbAs.GFP- or Ad.SERCA2a.GFP-treated myocytes.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Time to 50% relaxation (RT50) in myocytes from 9 matched preparations in which responses were compared in uninfected (Con), Ad.PlbAs.GFP-infected, and Ad.SERCA2a.GFP-infected myocytes. Data from 45-50 myocytes per preparation were measured, and statistical analysis was performed by ANOVA on pooled data. Significantly different from the respective control: *P < 0.05 and **P < 0.01; significantly different from respective basal levels: #P < 0.01 and ##P < 0.001 (Fisher's test). Bottom: example traces.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of the decrease in RT50 with isoproterenol in uninfected (Con; n = 7), Ad.PlbAs.GFP-treated (n = 12), and Ad.SERCA2a.GFP-treated (n = 8) myocytes.

Effects of thapsigargin on contraction and -adrenoceptor response. Thapsigargin significantly lengthened relaxation time and abolished the differences in basal amplitude between the control, SERCA2a-overexpressing, and low-phospholamban groups (Fig. 6), confirming the SR dependence of the adenoviral effects. However, isoproterenol was still able to reduce RT₅₀ significantly in the thapsigargin-treated myocytes. The positive inotropic effect of isoproterenol was unaffected by the presence of thapsigargin (Fig. 7).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   RT50 effect of 1 µM isoproterenol on myocytes contracting in the presence of 1 µM thapsigargin (Thaps; n = 3 preparations). *P < 0.05 compared with respective control; #P < 0.05 compared with control + thapsigargin in the absence of isoproterenol.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of thapsigargin on the response of uninfected myocytes (cultured for 48 h) to isoproterenol. Myocytes were allowed to contract in 1 µM thapsigargin for 10 min and then challenged with increasing concentrations of isoproterenol in the presence of thapsigargin (). Control concentration-response curves () were constructed on untreated myocytes from the same preparation (n = 3 preparations).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study shows that even partial depletion of phospholamban can be almost as effective as marked overexpression of SERCA2a in increasing contraction amplitude and accelerating relaxation of adult rabbit myocytes. Neither produced effects that exceed the natural stimulation of either contraction amplitude or relaxation time by catecholamines. Inotropic responses to -adrenoceptor activation were unaffected by either SERCA2a overexpression or phospholamban downregulation, whereas lusitropy was accentuated, particularly at low catecholamine concentrations, and arrhythmogenic effects were unchanged or reduced.

The expectation of a supraphysiological effect of this strong SERCA2a overexpression was not realized in this study. SERCA2a overexpression per se was no more effective than isoproterenol at either increasing contraction or accelerating relaxation. In the presence of isoproterenol, the final RT₅₀ values for SERCA2a-overexpressing myocytes were similar to those for phospholamban-depleted cells and only 25 ms faster than for uninfected myocytes. This increment is minor compared with the 110-ms reduction by isoproterenol alone. This has been the experience also in transgenic mice, with acceleration of relaxation at modest levels in SERCA2a overexpressors, sometimes even less than those observed in Plb-KO transgenic mice (1, 20). Conversely, phospholamban depletion was unexpectedly effective. The near equivalence between phospholamban downregulation and SERCA2a overexpression was surprising given previous disappointing reports with phospholamban antisense in adult myocytes (13).

The interaction between increased SERCA2a activity and -adrenoceptor stimulation differed for the inotropic, lusitropic, and arrhythmogenic effects. The positive inotropic effect of isoproterenol was not affected by either phospholamban depletion or SERCA2a overexpression, either in terms of maximum contraction amplitude or EC₅₀. This suggests that the increased Ca²⁺ uptake into the SR does not, per se, contribute to the inotropic effect of catecholamines if the basal contraction amplitude is not raised. In turn, this implies that that Ca²⁺ influx via the L-type Ca²⁺ channel, the other main source of activator Ca²⁺, predominates in the control of contractile force by -adrenoceptor agonists. The lack of alteration of the isoproterenol response by thapsigargin, which essentially removes the SR contribution to contraction, would support this contention. In Plb-KO mice the response of the myocytes to isoproterenol has been reported as decreased (35), but the difference from the present study is that, in the Plb-KO myocytes, isoproterenol is acting on a background of raised basal contraction. The maximum amplitude attained after isoproterenol treatment, however, is similar in cells from wild-type and Plb-KO mice, probably because of a intrinsic limitation in the maximum contraction of an individual myocyte. The possible increase that could be produced by each concentration of isoproterenol is therefore reduced because of these limits. In the present study we have selected a stimulation frequency at which basal contraction is only marginally affected by SERCA2a overexpression or phospholamban depletion. Under these circumstances there is no interaction between -adrenoceptor inotropy and modulation of SERCA2a activity. In a similar way, Serikov et al. (28) showed that reducing basal contractility in Plb-KO mice, by decreasing extracellular Ca²⁺, is able to restore responses to -adrenoceptor stimulation. Alternatively, the difference between our findings and those in transgenic animals may be related to recent reports that -adrenoceptor density can be decreased in Plb-KO mice (34).

With respect to acceleration of relaxation, phospholamban downregulation was not as effective as maximal -adrenoceptor stimulation in reducing RT₅₀, which would be expected because Ad.PlbAs.GFP reduced phospholamban levels by only 50%. Addition of isoproterenol to phospholamban-depleted myocytes was able to decrease RT₅₀ further. SERCA2a overexpression was slightly more effective than phospholamban depletion in accelerating relaxation and was approximately equal to the effect of maximum -adrenoceptor stimulation. However, isoproterenol was able to decrease RT₅₀ significantly even in SERCA2a-overexpressing myocytes. This additional effect of isoproterenol over SERCA2a overexpression either could indicate some residual regulation by phospholamban or could be ascribed to other mechanisms of -adrenoceptor-dependent acceleration of relaxation such as phosphorylation of troponin I (16). Acceleration of relaxation by either SERCA2a overexpression or phospholamban downregulation was abolished after depletion of the SR Ca²⁺ stores by allowing myocytes to contract in thapsigargin, confirming the SR dependence of the effect. Isoproterenol was able to decrease RT₅₀ even in this SR-independent mode, which again suggests the existence of non-SR-related mechanisms to accelerate relaxation. In Plb-KO mice, SR-independent stimulation of relaxation by catecholamines was seen in muscle strips (18, 23) and accounted for <20% of the total acceleration, in agreement with the present study. Troponin I is the likely mediator of this effect, because transgenic mice lacking the PKA phosphorylation site on troponin I show reduced relaxation responses to -adrenoceptor stimulation (24). Crossing mice with mutant or slow skeletal troponin onto a phospholamban-null background eliminated the lusitropic effect of -adrenoceptor stimulation completely, confirming that phospholamban and troponin I are the two main PKA substrates mediating enhanced relaxation (24, 34).

Although -adrenoceptor-mediated inotropy was unaffected by SERCA2a overexpression or phospholamban depletion, sensitivity to the lusitropic effects of catecholamines was altered. Isoproterenol is acting on a background of accelerated relaxation (decreased RT₅₀) in either SERCA2a-overexpressing or phospholamban-depleted myocytes. The minimum RT₅₀ value after isoproterenol treatment was, however, not very different among control, SERCA2a-overexpressing, and phospholamban-depleted myocytes. The possible effect of each concentration of isoproterenol was therefore reduced, and the slope of the concentration-response curve was shallower. There was also a shift in the concentration-response curve to lower values, with sensitivity increased ~0.5 log unit for SERCA2a overexpression and >1 log unit for phospholamban downregulation. This may have consequences for in vivo cardiac function, because an increase in the lusitropic effect for a given increase in contraction might be beneficial. We showed previously (12) that myocytes from failing heart, while relatively insensitive to the inotropic effects of catecholamines, are actually more sensitive to the acceleration of early relaxation (RT₅₀).

Paradoxically, although early relaxation is accelerated by catecholamines in the failing human heart, there can often be a slowing of the late relaxation phase. This phase is often already pronounced in human cells and can be converted to early aftercontractions by -adrenoceptor stimulation (33). The effect is likely to be related to reactivation of the sarcolemmal L-type Ca²⁺ channel during the action potential (36). Catecholamines can both increase action potential duration (19) and increase the probability of channel reopening (27), either of which could contribute to the likelihood of early afterpotentials that occur before repolarization and are sufficient to generate aftercontractions (3). We showed previously (4) that SERCA2a overexpression in rabbit myocytes decreases the incidence of aftercontractions and accelerates RT₉₀ (late relaxation) as well as RT₅₀. Results from the present study suggest that phospholamban downregulation has a similar effect. Acceleration of RT₉₀ was also observed with Ad.PlbAs.GFP treatment of human myocytes (6). In that study and in the present study, there is evidence that increasing SERCA2a activity (by SERCA2a overexpression or phospholamban depletion) can additionally reduce or suppress isoproterenol-mediated aftercontractions.

At high isoproterenol concentrations, contraction in the myocytes is frequently disrupted by arrhythmias indicating a Ca²⁺ overload state and characterized by disorganized contraction, loss of synchronization with the stimulation pulse, and waves of contraction likely resulting from spontaneous Ca²⁺ release from the SR. These arrhythmias have more in common with delayed afterdepolarizations in their occurrence and Ca²⁺ dependence (3). The incidence of these arrhythmias at isoproterenol concentrations of 1 µM was slightly lower in SERCA2a-overexpressing or phospholamban-deficient myocytes. This indicates that the higher SR Ca²⁺ load in these myocytes has not made the cells more sensitive to the arrhythmic effects of -adrenoceptor stimulation.

The spectrum of changes observed with the adenoviral vectors used in the present study would be predicted to be favorable for the failing human heart because it includes reversal of the depressed contractile response to increasing stimulation frequency (5), increased ability of catecholamines to accelerate the slowed relaxation (9), and reduction of catecholamine-induced aftercontractions, which underlie the characteristic torsades de pointes arrhythmia (25). Interest in stimulation of SERCA2a activity as a possible strategy for gene therapy of the failing myocardium has been generated by the beneficial effects in animal models of cardiomyopathy and in myocytes from diseased human hearts (6, 8, 11, 15, 21, 22, 26, 29, 31). Reduction of the inhibitory action of phospholamban is an attractive option because of the small size of the cDNA inserts needed for antisense/dominant-negative phospholamban constructs, making it suitable for use with the newer generation of adeno-associated viruses (AAV). Infection with AAV is less immunogenic that with adenovirus and has proved to be longer lasting in the myocardium (7). Our demonstration that partial phospholamban depletion can be as effective as SERCA2a overexpression supports the therapeutic use of this strategy.

In conclusion, functional effects on contraction and early relaxation of increased SERCA2a activity are similar to those of -adrenoceptor stimulation in both direction and magnitude. SERCA2a overexpression is only slightly more effective than partial phospholamban downregulation and does not have a greater propensity for supraphysiological increases in contraction amplitude or acceleration of relaxation. Isoproterenol-induced increases in contraction are not affected by increased SERCA2a activity, but relaxation becomes more sensitive to -adrenoceptor stimulation. There is no evidence in the study for any deleterious interaction between catecholamines and gene transfer-mediated stimulation of SERCA2a activity in terms of arrhythmia generation.