Cardiovascular Research

Cardiovascular Research 2001 49(2):288-297; doi:10.1016/S0008-6363(00)00234-0
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling

Nathalie Chossat^a, Frank Griscelli^b, Philippe Jourdon^c, Damien Logeart^a, Thierry Ragot^b, Michèle Heimburger^a, Michel Perricaudet^b, Anne-Marie Lompré^c, Stéphane Hatem^a and Jean-Jacques Mercadier^a,*

^aINSERM U 460, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France
^bCNRS UMR 1582, Institut Gustave Roussy, Villejuif, France
^cCNRS URA 1131, Université de Paris XI, Orsay, France

^* Corresponding author. Tel.: +33-1-4485-6158; fax: +33-1-4485-6157 jjmercadier{at}wanadoo.fr

Received 3 March 2000; accepted 24 August 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: We examined the functional consequences of expressing adult rabbit fast skeletal sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA1a) in isolated adult rat ventricular myocytes. Methods: Myocytes were infected with a recombinant adenovirus harboring SERCA1a. Then 2 days after myocyte infection, protein expression was estimated using Western blot and SDS–PAGE analysis. We also measured the ATP-dependent oxalate-facilitated Ca²⁺ uptake of myocyte homogenates and monitored Ca²⁺ transient in myocytes loaded with the Ca²⁺ dye, indo-1. Results: SERCA1a gene expression resulted in a 36% increase in the total SERCA protein level in infected myocytes compared to controls (P<0.01), while SERCA2 and phospholamban levels did not change. This increase was associated with a 42% rise in SR Ca²⁺ uptake (P<0.01), while (the time constant of Ca²⁺ transient decay), and the time to peak fell by 32% (P<0.01) and 38% (P<0.001), respectively. Increasing the frequency of stimulation from 0.2 to 2 Hz decreased in both cell types (P<0.01). However, the decrease was much smaller in infected (P<0.01) than in uninfected cells (P<0.001). Isoproterenol (1 µM) further decreased in infected myocytes by 23% (P<0.05). In these cells, the diastolic [Ca²⁺]_i decreased by 50% (P<0.05) while the systolic [Ca²⁺]_i increased by 19% (P<0.05). No difference was found in the speed of SR Ca²⁺ reloading after caffeine washout between the two cell types. Conclusion: Adenovirus-mediated SERCA1a gene transfer to adult rat ventricular myocytes enhances SR Ca²⁺ handling to a degree similar to that observed following physiological stimulation.

KEYWORDS Gene therapy; Myocytes; Ca-pump; SR (function); Cell culture/isolation

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This article is referred to in the Editorial by A. Baartscheer (pages 249–252) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) plays a major role in the regulation of intracellular calcium cycling in muscle cells. SERCA is a 110-kDa transmembrane protein that pumps two molecules of Ca²⁺ from the cytoplasm into the SR per molecule of ATP hydrolyzed, a mechanism largely responsible for muscle relaxation. The three SERCA genes so far identified (SERCA1, SERCA2 and SERCA3) encode six distinct protein isoforms by alternative splicing [1]. SERCA2a is the isoform expressed in adult cardiac myocytes and SERCA1a is the adult fast skeletal muscle isoform. The SERCA isoforms differ mainly by their sensitivity to Ca²⁺, but SERCA1a and SERCA2a have similar Ca²⁺ transport rates in vitro [1].

Changes in SERCA2 gene expression have been observed in animal models of cardiac hypertrophy and failure, as well as in human cardiomyopathies [2,3]. Depressed SR Ca²⁺ uptake and decreased levels of SERCA2 mRNA have been consistently observed in most models of cardiac hypertrophy and failure, but changes in protein levels are controversial [4,5]. Nonetheless, there is general agreement that the SERCA protein level falls in markedly hypertrophic ventricles and is further decreased in failing ventricles [2]. These changes are of major pathophysiological importance, as they may be responsible, at least in part, for the changes in intracellular calcium cycling and myocardial relaxation that occur in the failing myocardium, suggesting that SERCA gene transfer might rescue the compromised SR and contractile muscle function.

There has been great interest in ways of inducing SERCA gene overexpression in cardiac myocytes, focusing on isolated myocytes in vitro or the transgenic approach and gene transfer technologies in vivo [6–12]. Adenovirus-mediated SERCA2 overexpression in isolated neonatal rat ventricular myocytes increases SR Ca²⁺-ATPase activity, intracellular calcium transient and myocyte shortening [13]. SERCA2 gene transfer also restores depleted SERCA protein levels and shortens the prolonged Ca²⁺ transients resulting from a PMA (phorbol-12-myristate-13-acetate)-induced decrease in SERCA2a gene expression [7]. These results are consistent with those observed in transgenic mice overexpressing SERCA isoforms [10,11]. Recently, SERCA2a overexpression in myocytes isolated from failing human ventricles was shown to improve intracellular Ca²⁺ handling and to increase contraction and relaxation velocities [14], while SERCA2a gene transfer in vivo improved left ventricular function in rats in transition to heart failure [12]. Taken together, these studies show the value of SERCA gene transfer for our understanding of the role of the encoded protein in the function of cardiac myocytes, and of the feasibility of gene therapy of heart failure. However, in vitro SERCA gene transfer has often been applied to neonatal myocytes, in which SR function is immature, and little attention has been paid to the relationship between the level of SERCA protein expression, SR function and the resulting physiological effect. We therefore used adenoviruses to express the rabbit SERCA1a isoform — which can readily be discriminated from the endogenous SERCA2a isoform — in mature myocytes isolated from the ventricles of adult rats, and examined the consequences of SERCA1 protein expression on SR Ca²⁺ uptake, the Ca²⁺ transient and its alterations following an increase in the rate of stimulation and exposure to the β-adrenergic agonist isoproterenol.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Construction and purification of recombinant adenoviruses
The E1-defective adenovirus Ad-CMVSERCA1a, expressing the SERCA1a gene under the control of the cytomegalovirus (CMV) immediate-early promoter, was constructed as follows. The plasmid pAd.CMV.SERCA1a was obtained after subcloning a SpeI-EcoRV fragment (3.06 kb) from the pBSK.SERCA1a plasmid containing the full-length rabbit SERCA1a cDNA (a gift from Moutin [15]) into the pAd.CMVIcpA cleaved by NheI and EcoRV. This plasmid carries the first 0.45 kb of the Ad5 genome left extremity, the CMV enhancer/promoter followed downstream by a chimeric intron (the sequence of which is derived from the pCI plasmid, Promega), the SERCA1a gene flanked at its 3' end by the sequence containing the bovine growth hormone polyadenylation signal (obtained from plasmid pcDNA3, Invitrogen) and a 3.2-kb viral genome sequence from the region downstream of the E1 gene. The recombinant virus, designated Ad.CMV.SERCA1a, was obtained by homologous recombination after transfection of 10 µg of plasmid pAd.CMV.SERCA1a DNA and 5 µg of the large ClaI fragment (2.6–100 map units) of Ad5-dl324 viral DNA in the 293 cell line. The cells were overlaid with agar and incubated for 10 days; viral plaques were then isolated on 293 cells and amplified twice before DNA extraction. SalI and EcoRI restriction analysis confirmed the identity and clonality of the recombinant adenovirus, which was further purified once before stock preparation on 293 cells. The virus preparations obtained after lysing infected cells were purified twice in a CsCl density gradient (to avoid carrying over impurities from the cell culture) and extensive dialysis against saline buffer supplemented with 10% glycerol (v/v); aliquots were stored at –80°C. Viral stocks were titrated by plaque assay on 293 cells.

2.2 Cardiac myocyte isolation, culture and adenoviral infection
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult male Wistar rat ventricular myocytes were isolated as previously described [16]. Cells were infected on the day of isolation, 1 h after plating, with Ad.CMV.SERCA1a at 50 or 100 plaque-forming units of virus per cell (p.f.u./cell) for 1 h. The medium was then renewed and the cells were maintained in culture at 37°C for 48 h before further analysis.

2.3 Preparation of SR-enriched microsomes and cell homogenates
SR-enriched microsomes were prepared essentially as described by Levitsky et al. [17]. Briefly, myocytes were resuspended at 4°C in extraction medium containing 20 mM Tris–HCl (pH 7.4), 5 mM sodium azide and 30% glycerol, then disrupted with a P21 glass homogenizer (Polylabo). The homogenates were centrifuged at 1250xg for 2 min to remove cell debris. The supernatant (S1) was collected and centrifuged at 10 000xg for 5 min. The resulting supernatant (S2) was centrifuged at 100 000xg for 30 min to pellet the membranes. The pellet was resuspended in 50 µl of extraction medium then frozen in liquid nitrogen and stored at –80°C until use. Samples of left ventricle and extensor digitorum longus (EDL) muscle from a 300-g male Wistar rat were used as controls for SERCA2 and SERCA1, respectively. Frozen tissue samples were pulverized in liquid nitrogen, then homogenized and processed as described above.

2.4 Identification, localization and quantification of SERCA proteins
Two monoclonal antibodies (Affinity BioReagents) were used to characterize and quantify SERCA1 and SERCA2 isoforms. The anti-SERCA2, raised against purified canine cardiac SR vesicles, was shown not to cross-react with SERCA1 in a broad range of species. The anti-SERCA1, raised against purified rabbit skeletal muscle, was shown to specifically recognize SERCA1 in a wide range of species. A third monoclonal antibody, raised against phospholamban (PL), purified from canine cardiac sarcoplasmic reticulum (Upstate Biotechnology), was used to quantify PL.

In a first series of experiments, anti-SERCA1 was used to reveal SERCA1 expression in infected myocytes and to determine the virus concentration (MOI) required for optimal gene transfer efficiency and SERCA protein expression. Briefly, 48 h after infection, cells were fixed in 3.7% formaldehyde at room temperature for 10 min. Endogenous peroxidase was saturated with immunopure peroxidase suppressor (Pierce) for 30 min at room temperature and cells were permeabilized on ice in 0.2% Triton X-100/PBS (phosphate buffered saline) for 15 min. Non-specific sites were blocked with a solution containing 5% BSA in PBS for 30 min before incubating the cells with anti-SERCA1 diluted 1:400 at room temperature for 1 h. This was followed by 1-h incubation with a 1:100 dilution of peroxidase-conjugated anti-mouse IgG to count the number of positive myocytes. Antibody binding was visualized by exposing the cultures to the DAB substrate (Pierce kit) for 1–5 min. We used confocal microscopy for precise SERCA1a localization within myocytes. Cells were incubated with a 1:250 dilution of anti-SERCA1 antibody at room temperature for 1 h. After washing, cells were incubated with a 1:400 dilution of FITC conjugated anti-mouse IgG (Molecular Probes) for 90 min. Images were taken using a Leica TDS-4D confocal laser-scanning microscope (Leica, Heidelberg, Germany). SERCA1 was visualized with a fluorescent isothiocyanate (FITC)-fluorescent set: excitation 488 nm, beam splitter 518 nm, emission band-pass filter appropriated for FITC.

To determine the total amount of SERCA protein expressed in infected myocytes (endogenous SERCA2 plus exogenous SERCA1), proteins in homogenates purified from infected and uninfected myocytes were separated on SDS–PAGE as previously described [17]. Briefly, SDS–PAGE was performed on a 7.5% separating gel with a 3% stacking gel and proteins were stained with SYPRO^® Red (Molecular Probes). Gels were scanned with the Molecular Dynamics Storm^® and band densities were assessed using ImageQuant^® software. Signals from protein bands of interest were normalized by dividing arbitrary fluorescence units by those of a reference band (dotted arrow in Fig. 2). Serial dilutions of the homogenate preparations showed a linear relationship between the amount of protein loaded and the fluorescence intensity. Accordingly, the ratios did not change with the amount of protein loaded. The identity of the SERCA band was determined by Western blot analysis. SDS–PAGE was performed as described above. The membranes were incubated with 1:1000 anti-SERCA2 or 1:2500 anti-SERCA1 antibody mixed with 1:5000 anti-phospholamban antibody to study PL gene expression. A 1:10 000 dilution of peroxidase-conjugated anti-mouse IgG (Pasteur Diagnostic) was used as the secondary antibody. Immunoreactivity was determined using the enhanced chemoluminescence (ECL) reaction (NEN).

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Detection of SERCA1a protein with a specific antibody in the same culture of uninfected myocytes and myocytes infected by the recombinant adenovirus Ad.CMV.SERCA1a at an MOI of 50 p.f.u./cell. (A) Uninfected myocytes 48 h after isolation. (B) Infected myocytes 48 h after infection; bar=50 µm. (C) Localization of SERCA1a in an infected myocyte using confocal microscopy; bar=12 µm.

 
2.5 Ca²⁺ uptake assay
Ca²⁺ uptake activity was measured on myocyte homogenates at 30°C in 4.6 ml of medium containing 100 mM KCl, 6 mM MgCl₂, 0.2 mM EGTA, 5 mM sodium azide, 30 mM Tris–HCl buffer, 5 mM Mg-ATP, 15 mM K-oxalate (pH 7.0) and 125 µM ⁴⁵CaCl₂, which resulted in a free calcium concentration of 0.57 µM. The reaction was started by adding an aliquot of homogenate containing 37.5 µg of protein. To determine the Ca²⁺ uptake rate, 0.4-ml aliquots were taken from the same reaction tubes at various times (0.5–10 min) and the reaction was stopped by adding a washing solution containing 100 mM KCl, 10 mM histidine (pH 6.4), 1 mM EGTA. The mixture was filtered through GF/C Whatmann filters and the filters were washed three times with 5 ml of washing solution before counting in a liquid scintillation counter. The Ca²⁺ uptake activity of each sample was measured in triplicate. Because of the large amount of myocyte homogenate required for each assay, only the pCa (–log [Ca²⁺]) of 6.25 was studied. This value corresponded to a free calcium concentration of 0.57 µM, which is close to the K_d (constant of dissociation) of the enzyme. The free calcium concentration was calculated according to Fabiato and Fabiato [18]. The initial rates of ATP-dependent oxalate-facilitated Ca²⁺ uptake were calculated by least-squares linear regression analysis of Ca²⁺ uptake values at 30, 60, 90 s and 10 min. The results were analyzed using Microcal Origin software.

2.6 Measurement of calcium transients
Cells were loaded with 10 µM of the permeant indo-1 AM Ca²⁺ dye (Interchim) added to the culture medium in the presence of 0.5 mg/ml pluronic acid (Sigma) at room temperature for 15 min. Pluronic acid was used to improve indo-1 dispersion and facilitate cell loading. The cells were then superfused with Tyrode solution containing (in mM): 136 NaCl, 5.4 KCl, 2 CaCl₂, 1.06 MgCl₂, 0.33 NaH₂PO₄, 2 CaCl₂, 5 KCl and 10 HEPES, pH adjusted to 7.4 with NaOH for at least 40 min to wash out extracellular dye and permit intracellullar indo-1 de-esterification. The myocytes were stimulated by field electrodes at a default rate of 0.5 Hz (500-ms duration at suprathreshold) and excited at 340 nm with a xenon UV lamp. Emission was measured at 405 and 480 nm (F405, F485) using two photomultipliers attached to an inverted microscope (Nikon TDM, Diaphot). The cell was then stimulated for at least 1 min at 0.5 Hz in order to maintain stable SR Ca²⁺ loading. Calcium transients were expressed as ratios of indo-1 fluorescence at 405 and 485 nm (R = F405/F485) or as changes in free intracellular Ca²⁺ concentrations ([Ca²⁺]_i) using the following equation [Ca²⁺]_i=K_dβ/(R–R_min)/(R_max/R) where K_d is the dissociation constant of indo-1(213 nM), β is the ratio of fluorescence at 485-nm in the absence of [Ca²⁺]_i and at saturating [Ca²⁺]_i, R is the ratio of fluorescence at 405 and 485 nm (R = F405/F485), R_min and R_max are the ratios of indo-1 at 485 nm in the absence of Ca²⁺ and the presence of saturating Ca²⁺ concentration, respectively [19]. Determination of R_min and R_max was performed in indo-1 loaded cells treated with 3 µM carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP, Sigma) and 10 mM 2-deoxyglucose (Sigma) to achieve metabolic inhibition and to limit hypercontracture. To obtain R_max, 3 µM ionomycine (Calbiochem) was added to a solution containing 2 mM Ca²⁺ whereas to obtain R_min cells were incubated with a bath solution containing 0 mM Ca²⁺ and 5 mM EGTA (ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid) [20].

Isoproterenol (Sanofi Winthrop Pharmaceuticals) was used at a concentration of 10^–6 M and caffeine (Merck) at 1 mg/ml. Both compounds were dissolved in Tyrode solution containing 2 mM Ca²⁺ and 5 mM K⁺. The various compounds were applied via a rapid perfusion system to limit the time between applications. We focused on tau (), the time constant of the monoexponential decay of the Ca²⁺ transient using the equation: y(t)=A·e^–t/+B where t represents the continuous variable of time. The fits were analysed using GraphPad software. Only fits with a r²>0.95 were used.

2.7 Statistical analysis
Data are expressed as means±S.E.M. One- or two-way analysis of variance (ANOVA) or Student's t-test were used to test for statistical differences between infected and uninfected myocytes as appropriate. When ANOVA revealed significant differences, group-to-group comparisons were performed using the Scheffe F-test. A value of P<0.05 was assumed to reflect a significant difference.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 SERCA protein expression in myocytes infected with Ad.CMV.SERCA1a
To check the specificity of the two anti-SERCA antibodies, we performed Western blots using various muscle types and adult rat ventricular myocytes. As shown in Fig. 1A, the anti-SERCA2 antibodies detected a single sharp protein band of the expected apparent MW (110 kDa) in the rat ventricle and isolated myocytes. A band was also visible in the soleus muscle and diaphragm with longer exposure times (not shown), whereas no signal was visible in EDL. Conversely, the anti-SERCA1 antibodies revealed a single band of similar MW in EDL and diaphragm only.

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) Localization of SERCA1a transgene expression. Western blot analysis of SERCA1a and SERCA2 in homogenates of ventricle (V), diaphragm (D), EDL (E) and soleus (S), in the membranes of uninfected myocytes (NI mb) and infected myocytes (I mb), and in the cytosol of infected myocytes (I cyto). A total of 15 µg of each homogenate and 15 µg of isolated myocyte samples were loaded. (B) Western blot analysis of homogenates of uninfected and infected myocytes (2, 10 and 20 µg of protein were loaded).

 
To minimize the consequences of myocyte de-differentiation, which occurs rapidly in culture [21], we placed the SERCA1a cDNA under the constitutive CMV promoter, which yielded strong and rapid expression of the transgene. Indeed at an MOI of 50, almost all myocytes exhibited SERCA1a protein expression after 2 days in culture (Fig. 2B), whereas MOIs higher than 50 led to increased rates of cell death without increasing the transfection efficiency. To localize more precisely SERCA1a within infected myocytes, we used confocal microscopy (Fig. 2C). Immunofluorescent labeling of SERCA1 was homogeneously located throughout the cell, essentially distributed with a regularly spaced transverse band pattern but also as strands oriented longitudinally. No staining was detected in uninfected myocytes (not shown).

Western blot analysis of SR-enriched microsomes and cytosolic fractions revealed that anti-SERCA2 antibodies labeled a single protein band of the expected MW in SR-enriched microsomes from both infected and uninfected myocytes, whereas no signal was detected in the cytosolic fraction (Fig. 1A). The intensity of the SERCA2 signal was similar in infected and uninfected cells. The anti-SERCA1 antibodies gave a strong protein signal of the expected MW in the membrane fraction of infected myocytes, while no signal was detected in uninfected myocytes. In some experiments a weaker signal was also detected in the cytosolic fraction. Fig. 1B shows a typical Western blot of homogenate proteins from infected and uninfected myocytes probed with the two anti-SERCA antibodies plus the anti-PLB antibody. As in SR-enriched microsomes, SERCA2 was detected in homogenates of uninfected and infected myocytes, whereas SERCA1 was only detected in infected myocytes. The expression level of SERCA2 and PLB were not affected by SERCA1 expression. Together, these results indicated (i) that SERCA1a protein was expressed in infected myocytes, (ii) that it was appropriately addressed to the SR membrane, and (iii) that expression of the endogenous SERCA2a isoform and PLB was unaffected by expression of the transgene.

To assess the total amount of SERCA present in homogenates from uninfected and infected myocytes, various amounts of proteins were separated on SDS–PAGE and stained with SYPRO^® Red (Fig. 3). A similar band pattern was observed with the two types of homogenate, except for one protein band with an apparent MW of 110 kDa, the fluorescence intensity of which was stronger in infected than in uninfected myocytes. Normalization of the 110-kDa band signal to a reference band indicated a 36±9% increase in infected myocytes compared to uninfected myocytes (n = 6, P<0.01). Western blotting confirmed that this band corresponded to SERCA2 in uninfected myocytes and to both SERCA isoforms in infected myocytes (not shown).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Quantification of total SERCA protein in uninfected myocytes and in infected myocytes with SDS–PAGE (10, 20 and 30 µg of protein were loaded).

 
3.2 SR Ca²⁺ uptake in myocyte homogenates
To examine the effects of SERCA1 gene expression on SR calcium transport, we measured the ATP-dependent oxalate-facilitated Ca²⁺ uptake activity of infected myocyte homogenates. Fig. 4A shows a typical example of the time course of Ca²⁺ uptake in homogenates from infected (I) and uninfected (NI) myocytes. At the pCa of 6.25 (close to the K_d value of the enzyme) [22] Ca²⁺ uptake in control myocytes increased gradually with time and tended to reach a plateau at 10 min. In infected myocytes Ca²⁺ uptake increased much faster, reaching a value markedly higher than the control value at 10 min. The mean initial rate of Ca²⁺ uptake increased by 42% in infected myocytes compared to controls (54.66±3.61 vs. 38.46±3.33 nmol Ca²⁺/mg protein per min, n = 7 and 6, respectively, P<0.01, Fig. 4B). These results were consistent with an increased number of functional SR Ca²⁺ pumps in infected myocytes than in controls.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) Time course of oxalate-dependent calcium uptake in homogenates of uninfected myocytes (U) and infected myocytes (I). The control represents calcium uptake in the absence of both oxalate and ATP. (B) Initial rate of SR Ca2+ uptake (Vmax) in homogenates of uninfected myocytes (U) and myocytes infected with Ad.CMV.SERCA1a (50 p.f.u./cell) (I) at a pCA of 6.25.

 
3.3 Ca²⁺ transients
To examine whether SERCA1 expression and the resulting increase in total SERCA protein affected the SR cycling process, we measured Ca²⁺ transients in control and infected myocytes. Fig. 5A shows a typical example of Ca²⁺ transients recorded in an uninfected myocyte and a myocyte infected by Ad.CMV.SERCA1a. At a stimulation rate of 0.5 Hz, myocytes infected with Ad.CMV.SERCA1a had a mean 38% decrease in time to peak (39.6±1.5 vs. 63.6±3 ms, n = 25 and 19, respectively, P<0.001) and 32% decrease in (595±38 vs. 404±29 ms, n = 7 and 8, respectively, P<0.01) compared to controls reflecting the presence of functional SERCA1 protein in infected myocytes. Myocytes infected with Ad.RSV.lacZ had Ca²⁺ transients similar to those of uninfected myocytes (not shown). Increasing the frequency of stimulation from 0.2 to 2 Hz decreased in both cell types (P<0.001 and P<0.01 for uninfected and infected myocytes, respectively; Fig. 5B). However, whereas the decrease in averaged 51% in uninfected myocytes it was only 24% in infected myocytes (P<0.001). Stimulating myocytes at 4 Hz failed to trigger analyzable Ca²⁺ transients in the two cell types. Isoproterenol decreased by 18% in uninfected myocytes (n = 5, P<0.01 Fig. 5C) and by 23% in infected myocytes (n = 6, P<0.05). Taken together, these data indicated that Ca²⁺ transients in infected myocytes were still modulated by the β-adrenergic pathway.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 (A) Representative recording of the Ca2+ transient in indo-1-loaded uninfected myocytes and myocytes infected with Ad.CMV.SERCA1a (50 p.f.u./cell). (B) Effect of the frequency stimulation on the time constant ( of the Ca2+ transient decay) in uninfected (U) and infected myocytes (I). (C) Effect of isoproterenol stimulation on of uninfected (U) and infected (I) myocytes.

 
In a number of experiments, we measured the diastolic and systolic [Ca²⁺]_i in the two cell types (Table 1). In infected myocytes, systolic [Ca²⁺]_i increased by 19% while diastolic [Ca²⁺]_i decreased by 50% compared to uninfected cells. Accordingly, the amplitude of the Ca²⁺ transient was higher in infected than in uninfected cells (P<0.05). Of note was the very homogeneous values of diastolic [Ca²⁺]_i in infected compared to uninfected myocytes (see the S.E.M. values in Table 1). To precisely determine the degree of SR Ca²⁺ loading in infected and uninfected myocytes, we measured [Ca²⁺]_i in unstimulated myocytes before and after caffeine application. We found a 23% increase in the difference in [Ca²⁺]_i before and after caffeine application in infected compared to uninfected myocytes, confirming an increased SR Ca²⁺ load in the former (P<0.05).

View this table: [in this window] [in a new window]   Table 1 Alterations in intracellular free Ca2+ concentration ([Ca2+]i) following SERCA1a gene transfer to adult rat ventricular myocytesa

 
Finally, we compared the kinetics of Ca²⁺ transient recovery upon caffeine washout in infected and control myocytes. The Ca²⁺ transients were monitored in infected and uninfected myocytes by using a train of stimulations delivered at a rate of 0.5 Hz (Fig. 6A). Despite an increased rate of Ca²⁺ transient decay in infected myocytes, reflecting SERCA overexpression, the speed of SR Ca²⁺ reloading was similar in the two cell types. Fig. 6B shows the mean percentage recoveries of the Ca²⁺ transient as a function of the Ca²⁺ transient serial number following caffeine washout. Recovery of the Ca²⁺ transient tended to increase in infected myocytes compared to uninfected control cells, but the difference did not reach statistical significance (n = 5 and 6 for uninfected and infected myocytes, respectively).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Recovery of Ca2+ transients following caffeine washout. (A) SR loading kinetics; (B) percentage recovery of Ca2+. , uninfected myocytes; , infected myocytes. Ad.CMV.SERCA1a, 50 p.f.u./cell.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In the present study, we found that following adenoviral SERCA1 gene transfer to adult rat ventricular myocytes (i) the total amount of SERCA expressed is significantly enhanced, (ii) SERCA1 is homogeneously distributed throughout the cell body, (iii) is functionally active, (iv) and induces a shortening of the Ca²⁺ transient, an increase in SR Ca²⁺ load and a marked decrease in diastolic [Ca²⁺]_i.

A number of studies have examined the effects of SERCA overexpression in cardiac myocytes but only a few examined carefully the relationships between the amount of expressed protein and the resulting physiological effect. We examined SERCA expression levels in infected and control myocytes so that a direct comparison could be made between protein expression and SERCA function in the same preparations. Using SDS–PAGE analysis we found a 36% increase in total SERCA in infected myocytes. As no change in SERCA2 was observed, it is reasonable to assume that the increase in total SERCA protein was due to expression of SERCA1. In their pioneering study of adenovirus-mediated SERCA2 gene transfer to neonatal cardiac myocytes Hajjar et al. [13] found a 75% increase in total SERCA. The difference between this and our study may be explained by the fact that neonatal myocytes, owing to their low level of endogenous SERCA expression, may have a greater capacity to accumulate exogenous SERCA than adult myocytes. In some experiments we detected small amounts of SERCA1 in the cytoplasm of infected myocytes supporting the idea that the space available in SR membranes for exogenous SERCA may be limited in adult myocytes. Giordano et al. [7] found a 82% increase in SERCA protein levels in neonatal rat myocytes when SERCA protein expression has been previously decreased by PMA pretreatment, but this increase represented only a 10% increase over SERCA protein levels in control untreated myocytes [7]. More recently, Del Monte et al. [14] restored normal myocyte contractile function by transferring the SERCA2 gene to failing human ventricular myocytes, another situation involving decreased SERCA protein levels. They did not indicate the percent increase in total SERCA protein following gene transfer but the 68% increase in the ATPase activity of SR membranes of infected myocytes is in good agreement with the study of Giordano et al. [7].

Other studies of SERCA2 overexpression used the transgenic approach. He et al. [8] and Baker et al. [11] showed that SERCA2 protein levels increased by 20 and 30–50% in transgenic mice, respectively, compared with wild-type animals. Only Loukianov et al. [10] observed a huge increase in total SERCA protein (250%) in a single line of SERCA1 transgenic mice harboring the highest transgene copy number. Although the reason for the difference between the results of this study and those of in vitro studies is unclear, it is possible that the very long period available for transgene expression in the transgenic model allowed the SR to adapt and accept the excessive amount of SERCA protein, a phenomenon that could not have occurred during the 2 days of transgene expression in vitro. The discrepancy in the level of exogenous SERCA expression among the various studies also reflects the difficulty of precisely quantifying the amount of the expressed protein following gene transfer. In this context, our finding of a large and homogeneous expression of SERCA1 throughout the cell body of infected myocytes is a strong indication of the good efficiency of gene transfer. Taken together, the results of these studies and our own indicate that the level of exogenous SERCA protein expressed in SR membranes following adenovirus-mediated gene transfer to ventricular myocytes is moderate except when the level of endogenous SERCA is initially low.

The immunofluorescent data in Fig. 2 provide the first indication of the subcellular distribution of an exogenous SERCA protein in adult myocytes. We found that SERCA1 is distributed parallel to the t-tubular system as indicated by the clear striation pattern inside the cell, similar to the distribution pattern of the endogenous protein at the level of the SR terminal cisternae [23]. We found also some degree of longitudinal immunolabelling in good agreement with previous reports [24]. Although the significance of this labeling is unclear, it may correspond to SERCA1 at the level of the longitudinal SR. Additional experiments using immunoelectron microscopy are necessary to clarify this point.

We found a 42% increase in the SR Ca²⁺ uptake rate in myocytes expressing SERCA1, a first indication that the transferred SERCA1 was functional. This increase was of the same order of magnitude as the 36% increase in the total SERCA protein level. Since we observed no change in the level of PL protein, it seems reasonable to infer that the total SERCA to PL ratio increased slightly in infected myocytes, possibly explaining why the increase in the SR Ca²⁺ uptake rate was slightly larger than that in total SERCA protein. We can also assume that the PL still exerted its physiological inhibitory effect on the Ca²⁺-ATPase function of SERCA in infected cells, as isoproterenol still increased the rate of Ca²⁺ transient decay. However, isoproterenol stimulation decreased similarly in control and SERCA1-transfected myocytes (18 and 23%, respectively), although the decrease should theoretically have been smaller in the latter because of the higher SERCA to PL ratio.

The most direct experimental approach to Ca²⁺ cycling process and the function of SR Ca²⁺ pumps in infected myocytes is the measurement of Ca²⁺ transients. Despite the fact that 2 days of culture may have induced some degree of cell dedifferentiation [21], the general mechanisms of EC coupling are preserved [7,9,13,14]. We found that the SERCA1 gene transfer resulted in a markedly shortened Ca²⁺ transient in infected compared to uninfected cells, mainly because of an acceleration in its relaxation phase and also because of a shorter time to peak. The acceleration in the relaxation phase of the transient is probably the most direct physiological effect of SERCA overexpression and has been observed in most studies [7,8,10,11,13]. Our 32% decrease in is in good agreement with most in vitro and transgenic studies that showed a Ca²⁺-transient 20–40% shorter in SERCA-transfected myocytes compared to controls, whatever the parameter used to characterize the Ca²⁺ transient (, the rate of transient decay, or t_1/2 or t₈₀, the time required to reach 50 and 80% of the decay, respectively). Only mice with massive SERCA1a protein overexpression and a 70% increase in the SR Ca²⁺ uptake rate exhibited a somewhat shorter Ca²⁺ transient (52% in t₈₀) [10] than that found in other studies. As could be expected, increasing the rate of myocyte stimulation decreased , and the decrease was larger in uninfected than in infected myocytes. This is probably due simply to the fact that the latter started from much lower values than the former, the two cell types converging towards a similar value around 350 ms at a stimulation rate of 2 Hz, a value that may represent the fastest rate of Ca²⁺ transient decay achievable in our experimental conditions. Indeed, higher stimulation rates failed to trigger analyzable Ca²⁺ transients because of both decreased transient amplitude and increased diastolic [Ca²⁺]_i values (not shown). The interpretation of the 38% shorter time to peak of the Ca²⁺ transient in transfected cells is more complex. The density of ryanodine receptors (RyR) was found to be unchanged in a previous study of myocytes with SERCA overexpression [8] and it is unlikely that ICa-L changes in infected cells. Accordingly, the most likely explanation for the shorter time to peak of the Ca²⁺ transient is that it is due to the increased Ca²⁺ gradient between SR and the cell cytoplasm, thus facilitating the extrusion of Ca²⁺ from SR upon cell stimulation. Our data showing a larger amplitude of the Ca²⁺ transient together with the larger caffeine-induced increase in [Ca²⁺]_i reflecting an increased SR Ca²⁺ load in infected myocytes compared to controls, support this hypothesis. It could be due also to a shorter time to reload SR and/or to recirculate Ca²⁺ from the site of Ca²⁺ uptake to that of Ca²⁺ release. However, the latter hypothesis is countered by the fact that the recovery of the control Ca²⁺ transient after caffeine washout was not significantly faster in infected myocytes than in controls. Alternatively, this could simply indicate the integrity in the trans-sarcolemmal mechanisms controlling the degree of SR Ca²⁺ repletion rather that the inability of overexpressed SERCA to fill SR with Ca²⁺ more rapidly. Additional experiments are required to clarify these various possibilities.

It is interesting to note that whereas uninfected cells exhibited scattered diastolic [Ca²⁺]_i values, most if not all infected myocytes demonstrated very homogeneously low values. This suggests that the main effect of SERCA overexpression — and consequently one of the main physiological roles of SR — is to control diastolic [Ca²⁺]_i and maintain it close to an optimal level. This could be of special importance in the context of gene therapy of heart failure, a situation in which diastolic [Ca²⁺]_i is increased [25] and many patients suffer sudden death, an outcome likely related to the occurrence of delayed afterdepolarizations due to the conjunction of high diastolic [Ca²⁺]_i levels and illegitimate SR Ca²⁺ releases. It remains to be determined if SERCA gene transfer will be able to reverse these deleterious aspects of Ca²⁺ handling in failing myocytes.

In summary, our results show that SERCA overexpression in adult ventricular myocytes induces alterations in cellular Ca²⁺ handling similar to those induced by an increased rate of cell pacing or β-adrenergic receptor stimulation. Together with recent studies using myocytes isolated from failing human hearts [14] and a rat model of heart failure [12] our results showing a ‘normalizing’ effect of SERCA overexpression on myocyte Ca²⁺ handling, provide additional evidence that SR functional improvement by SERCA gene transfer is a relevant target for innovative therapeutic approaches to heart failure.

Time for primary review 24 days.

	Acknowledgements

 
This work was supported in part by a grant from Rhone Poulenc Rorer. Nathalie Chossat was supported by grants from the Association Française contre les Myopathies and from the Institut Electricité Santé. Damien Logeart was supported by the Fondation pour la Recherche Médicale and the Caisse Autonome Nationale d'Assurance Maladie/Assistance Publique - Hôpitaux de Paris. The authors thank Caroline Vigne for her assistance in confocal microscopy.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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