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Am J Physiol Heart Circ Physiol 291: H1773-H1779, 2006. First published June 9, 2006; doi:10.1152/ajpheart.00411.2006
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Percutaneous intracoronary delivery of SERCA gene increases myocardial function: a tissue Doppler imaging echocardiographic study

Damien Logeart,1,2 Laurent Vinet,1 Thierry Ragot,3 Michèle Heimburger,1 Liliane Louedec,1 Jean-Baptiste Michel,1 Brigitte Escoubet,4 and Jean-Jacques Mercadier1

1Institut National de la Santé et de la Recherche Médicale U698, Bichat Hospital; 2Assistance Publique-Hôpitaux de Paris, Lariboisière Hospital, Department of Cardiology, Paris; 3Centre Nationale de Recherche Scientifique UMR 1582, Institut Gustave Roussy, Villejuif; and 4Institut National de la Santé et de la Recherche Médicale U722, Assistance Publique-Hôpitaux de Paris, Bichat Hospital, Paris, France

Submitted 20 April 2006 ; accepted in final form 16 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to examine the efficiency of adenovirus-mediated overexpression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a) gene in a realistic model based on percutaneous intracoronary delivery and on noninvasive functional monitoring. Catheter-based selective coronary delivery of saline or adenoviruses (Ad.CMV.SERCA1a or Ad.CMV.lacZ, 1010 plaque-forming units) was performed in the circumflex artery of rabbits. Effects were assessed and compared by using serial Doppler echocardiography, hemodynamics, and measurements of SERCA protein and Ca2+ uptake activity. On day 3, a 21% increase in SERCA proteins and a 37% increase in the maximal rate of Ca2+ uptake were observed in the transfected left ventricular (LV) walls of Ad.CMV.SERCA1a rabbits. Baseline hemodynamics and conventional echographic measurements of global LV function were poorly affected. In contrast, tissue Doppler imaging (TDI) was able to assess a strong increase in the baseline function of transfected LV walls, as assessed with maximal wall velocities (+32% and +43%, respectively) and strain rates (+18% and +30%, respectively). TDI parameters were closely related to the maximal rate of Ca2+ uptake (r2 = 0.68 for the systolic strain rate). Serial TDI analysis during follow-up showed that the effects lasted for 7 days and were no longer detectable 15 days after adenoviruses injection. In conclusion, LV function can be increased by adenovirus-mediated overexpression of SERCA in a clinically relevant model, and TDI provides an accurate and noninvasive tool for monitoring effects on global as well as regional myocardial function.

gene therapy; ventricular function; sarco(endo)plasmic reticulum Ca2+-ATPase; echocardiography

HEART FAILURE is a growing public health concern and is only partly mitigated by current drug therapy. The poor prognosis of specific subsets of patients has stimulated the search for novel therapeutic approaches. One candidate is gene-based therapy, but a number of concerns need to be overcome, including vector efficiency and safety, transgene regulation, and vector route delivery. Various vector delivery methods have been used (11–13, 15, 20–22, 31). Candidate gene therapies need to be validated in relevant animal models, including delivery strategies that could be applied to humans. Recently, we tested a percutaneous catheter-based approach to selective intracoronary delivery in adult rabbits. We showed that the procedure involving an increase in pressure and adenovirus residence time within the coronary vessels is more efficient than an increase in vessel wall permeability induced by drug pretreatment alone (24).

Failing cardiac muscle exhibits altered intracellular Ca2+ handling, which is closely related to muscle contraction and relaxation. Changes in Ca2+ handling by cardiac myocytes include defects in sarcoplasmic reticulum (SR) Ca2+ release and reduced Ca2+ uptake, with downregulation of sarco(endo)plasmic Ca2+-ATPase (SERCA2) and/or increased SERCA inhibition by phospholamban (9). Available drugs cannot alter Ca2+ uptake. Adenovirus-mediated SERCA overexpression in cardiomyocytes isolated from the left ventricle (LV) of patients with end-stage heart failure restored normal contractility (5). Cardiac SERCA overexpression (6, 27) and phospholamban inhibition (14, 16) in rodents models of heart failure improve cardiac function and remodeling and, possibly, survival (6). Approaches targeting Ca2+ handling now need to be studied in models more relevant to human in vivo gene delivery.

Here we examined the efficiency of adenoviral SERCA1a gene transfer by percutaneous catheter-based myocardial delivery to adult rabbits and validated in vivo tissue Doppler echocardiography for monitoring regional muscle function changes compared with hemodynamics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant adenoviruses. The E1-defective adenovirus Ad.CMV.SERCA1a, expressing the rabbit SERCA1a isoform under the control of the immediate and early promoter of the cytomegalovirus (CMV), was constructed as previously described (4). Enzymatic properties of the skeletal muscle isoform (SERCA1a) are very close to the cardiac SERCA2a isoform (17). The Ad.CMV.lacZ, containing a nuclear-targeted beta-galactosidase gene driven by the CMV promoter, was constructed with similar procedures and was used as control. Titers of virus stocks were determined by plaque titration on the 293 cell line and expressed as plaque-forming units (pfu). Viral particles to pfu ratios were 40 and 18 for Ad.CMV.SERCA1a and Ad.CMV.lacZ, respectively. Virus stocks were aliquoted in small volumes and stored in PBS with 10% glycerol at –80°C until use. All experiments were carried out using aliquots of the same virus stocks.

In vivo gene transfer to myocardium and study design. The study was approved by the French Accreditation for Laboratory Animal Care (authorization no. 00577). Adult New Zealand White rabbits, weighing 3.5–4 kg, were sedated with pentobarbital sodium (10 mg/kg iv), intubated, and mechanically ventilated. The right carotid artery and jugular vein were exposed. A 6-Fr sheath was placed in the jugular vein and a 4-Fr sheath in the carotid artery. Based on the results of our previous study (24), we used the protocol combining catheter-mediated occlusion of the coronary artery when adenoviruses were delivered downstream and, at the same time, retroinfusion of saline buffer into the coronary venous sinus to increase both the perfusion pressure and the residence time of the vector in the coronary vessel. This results in massive gene transfer within the inferior and lateral walls of the left ventricle. Briefly, coronary venous sinus catheterization was performed with a 5-Fr fluid-filled balloon catheter (Baxter), and complete occlusion was obtained by balloon inflation. A 3-Fr coronary catheter (Cook) was then placed in the left circumflex artery; coronary flow was interrupted for 20 s by occlusive engagement of the catheter, while 2 ml of saline (containing either saline buffer alone, 1 x 1010 pfu of Ad.CMV.SERCA1a or 1 x 1010 pfu of Ad.CMV.lacZ) was delivered downstream. Complete occlusion of the coronary venous sinus was performed simultaneously, and saline buffer was retroinfused.

Among 40 successfully operated and surviving animals, 24 (8 saline, 8 Ad.CMV.SERCA1a, and 8 Ad.CMV.lacZ) were euthanized on day 3 immediately after echocardiography and hemodynamics, and 16 (8 saline and 8 Ad.CMV.SERCA1a) were euthanized on day 15 after echocardiography and hemodynamics; in this last group, serial echography was performed on days 3, 7, and 15.

Echocardiography and tissue Doppler imaging. The rabbits were slightly sedated (spontaneously breathing, with pentobarbital sodium 7–10 mg/kg iv). Echocardiography was performed in the supine position with a Toshiba PowerVision 6000, SSA 370A, equipped with a phase array 7.5-MHz transducer. Data were transferred to a computer for off-line analysis (Ultrasound Image Workstation-300A, Toshiba). The LV was imaged in both parasternal long-axis and short-axis views at the papillary muscle level. LV fractional shortening (FS) was calculated from M-mode images, as (end-diastolic diameter – end systolic diameter)/end-diastolic diameter. Cardiac output (CO) was calculated as heart rate x [3.14 x (diameter of LV outflow)2/4] x aorta velocity-time integral. Peak velocities of systolic (S) and early diastolic (E) regional motions of the LV were measured by pulsed-wave tissue Doppler imaging (TDI) with the sample volume placed within the LV inferior wall from parasternal short-axis view (Sinf and Einf, Fig. 1B) and within the basal segment of the LV lateral and septal walls from apical view (Slat and Elat, and Ssep and Esep, respectively). Peak velocities of systolic- and early-diastolic motions were also measured by pulsed-wave TDI, with the sample volume placed at the lateral corner of the mitral annulus (Sann and Eann) to assess global LV function in its long axis. Strain rate of LV posterolateral wall was derived from time-movement color TDI and was measured from parasternal the short-axis view, as previously described (5). Briefly, velocity profiles were extracted from epicardial to endocardial layers by echo tracking; strain rate was computed off-line as the velocity gradient between the epicardium and the endocardium velocities, sampled every 2 ms and averaged over 5 to 10 cycles (Fig. 1A).


Figure 1
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  		Fig. 1. Follow-up of control and Ad.CMV.SERCA1a animals on days 3, 7, and 15 using tissue Doppler imaging (TDI) analysis. A: peak systolic and diastolic strain rates of posterolateral left ventricular (LV) wall measured by time-mode–color TDI. Saline ({square}, n = 8); Ad.CMV.SERCA1a ({blacksquare}, n = 8) are shown. B: peak systolic [Slat: saline ({triangleup}) and Ad.CMV.SERCA1a ({blacktriangleup})] and diastolic velocities [Elat: saline ({circ}) and Ad.CMV.SERCA1a (bullet)] of lateral LV wall measured by pulsed-wave TDI. *P < 0.01 vs. control. SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; CMV, cytomegalovirus.

 
Invasive hemodynamic study. Animals were sedated with pentobarbital sodium as described above. After subcutaneous anesthesia with 1% xylocaine, the carotid artery was exposed, and a 4-Fr sheath was then introduced. A 2-Fr micromanometer-tipped catheter (Millar, Houston, TX) was placed in the LV cavity. The LV pressure curve and derivatives were recorded with a MacLab apparatus to measure systolic, end-diastolic, and developed pressures and maximal and minimal first derivatives of pressure (dP/dtmax and dP/dtmin, respectively). All hemodynamic data were derived from the average of 15 steady-state cardiac cycles. After baseline measurements, animals received stepwise dobutamine infusions (1–10 µg·kg–1·min–1), and data were recorded at steady-state with each dose. To examine the effect of heart rate, a sheath was placed in the jugular vein, and atrial pacing was performed with 25-beats/min steps from baseline to 350 beats/min.

Expression of SERCA1 proteins and Ca2+ uptake. After euthanasia, the heart was excised, and the aortic root was immediately cannulated to retroperfuse the heart with cold buffer. Samples of various walls of the LV were immediately separated as previously described (24).

SERCA1 protein expression was determined immunochemically as previously described (25). Positive and negative controls were obtained from extensor digitorum longus (EDL) and noninfected hearts, respectively. Proteins in homogenates were separated on 4–20% SDS-PAGE (Cambrex, Rockland, ME). The membranes were incubated with 1:2,500 diluted monoclonal anti-SERCA1 antibodies (Affinity BioReagents). A dilution of 1:10,000 peroxydase-conjugated anti-mouse IgG (Pasteur Diagnostic) was used as secondary antibody. SERCA2 and beta-actin expression were also determined by using diluted monoclonal anti-SERCA2 (Santa Cruz Biotechnology) and anti-beta-actin (Sigma, St. Louis, MO) antibodies, respectively. Blots were scanned, and signals arising from protein bands of interest were normalized by dividing arbitrary fluorescent units by the amounts of protein loaded. Both SERCA1a and -2 signals were divided by beta-actin signals. The total amount of SERCA protein was determined with serial dilutions of 10, 20, and 30 µg of proteins from each homogenate. Homogenates from Ad.CMV.SERCA1a animals and from control hearts were loaded in 7.5% separating gels, and proteins were stained by Coomassie blue. Scanned signals arising from the 110-kDa SERCA bands were plotted against those of a reference band, as well as the concentration of loaded proteins. The linear relationship of signals was checked across serial dilutions from one homogenate when the ratios (SERCA to reference) did not change within the same homogenate.

SR-enriched microsomes were prepared as described by Levitsky et al. (23). Ca2+ uptake activity was measured as previously described (16). Briefly, it was measured at 30°C in 4.6 ml of medium containing (in mM) 100 KCl, 6 MgCl2, 0.2 EGTA, 5 sodium azide, 30 Tris·HCl buffer, 5 Mg-ATP, 15 K-oxalate (pH 7.0), and 125 45CaCl2, which resulted in a free Ca2+ concentration of 0.57 µM. Only the pCa of 6.25 was studied; this value corresponded to the free Ca2+ concentration of 0.57 µM, close to the Kd of the enzyme. 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 and 90 s. The Ca2+ uptake rate measured at 10 min, when the velocity of Ca2+ uptake was the slowest, was considered as the maximal capacity of Ca2+ uptake by the SR (total Ca2+ uptake).

Statistical analysis. Data are expressed as means ± SE. Comparison among the three groups was performed by ANOVA and two-by-two comparison with Bonferroni correction. Comparison of two means were performed by Student's test or paired t-test when necessary. Linear regression analysis was based on the least-squares method. P ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LV function as assessed with echocardiography. On day 3 after transfection, the LV size was similar in all the groups, as were the parameters of LV pump function (FS and CO) and the Doppler mitral inflow (E-to-A wave ratio and deceleration time of E wave) (Table 1). In contrast, TDI showed a marked increase in parameters of LV muscle function in its long-axis (motion of the mitral annulus) and regional function of transfected LV walls (inferior and lateral LV walls) in Ad.CMV.SERCA1a rabbits. Indeed, pulsed-wave TDI peak velocities were significantly increased compared with the control groups (22% and 32% increase in Sinf and Slat, respectively; and 17% and 43% increase in Einf and Elat, respectively), as well as systolic and diastolic peak strain rates (18% and 30%, respectively). There was no difference between Ad.CMV.lacZ- and sham-treated rabbits. The increase in TDI parameters lasted up to 7 days after Ad.CMV.SERCA1a injection and then declined; on day 15, Ad.CMV.SERCA1a animals were not different from control animals (Fig. 1). On the other hand, pulsed-wave TDI peak velocities of septum, i.e., nontransfected LV wall, were not significantly different in the Ad.CMV.SERCA1a group compared with the control groups on day 3 as well as days 7 and 15.


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  		Table 1. Echographic results on day 3

 
Invasive hemodynamic study. Table 2 shows hemodynamics measured on day 3 after saline or adenoviruses injection. Maximal and minimal dP/dt were slightly higher in Ad.CMV.SERCA1a rabbits (+19 and +18%, respectively) than in the other groups. Dobutamine infusion at 2.5 µg·kg–1·min–1 increased dP/dtmax in all groups (Fig. 2A). The effect was significantly greater in Ad.CMV.SERCA1a animals (6.5 ± 0.2 vs. 4.8 ± 0.2 and 5.0 ± 0.3 mmHg/s, P < 0.001). A similar effect was obtained with dP/dtmin (not shown). Dobutamine infusion at 10 µg·kg–1·min–1 resulted in a further increase in dP/dt reaching the same maximal value in all groups. To test the effect of heart rate on cardiac hemodynamics, the heart rate was increased in 25-beats/min steps by right atrial pacing; a significant increase in the dP/dtmax was observed only in the Ad.CMV.SERCA1a group, whereas this parameter remained unchanged in both control groups (Fig. 2B). On day 15, no difference was observed between Ad.CMV.SERCA1a and controls groups, including dP/dtmax under dobutamine infusion at 2.5 µg·kg–1·min–1 (5.2 ± 0.3 vs. 5.0 ± 0.4 and 4.7 ± 0.3 mmHg/s, P = not significant).


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  		Table 2. Hemodynamics obtained on day 3

 

Figure 2
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  		Fig. 2. A: LV maximal first derivative of pressure (dP/dtmax) and effects of dobutamine infusion (*P < 0.0001 vs. controls). B: LV dP/dtmax according to increase in heart rate during atrial pacing. Saline (white bar), Ad.CMV.lacZ (gray bar), and Ad.CMV.SERCA1a (black bar) are shown. *P < 0.05 vs. saline and Ad.CMV.lacZ; **P < 0.01 vs. saline and Ad.CMV.lacZ.

 
Expression of SERCA1 protein and Ca2+ uptake. Figure 3 shows a representative Western blot analysis of SERCA1a gene expression on days 3 and 15. Whereas no SERCA1a gene expression was detected in LV homogenates from saline and Ad.CMV.lacZ animals, significant levels of SERCA1a proteins were observed in LV homogenates from Ad.CMV.SERCA1a animals on day 3. According to the measurement of the total amount of SERCA protein, SERCA1a expression resulted in a 12% to 27% (21.1 ± 5.0%) overexpression of SERCA proteins in Ad.CMV.SERCA1a animals compared with control animals. SERCA1a proteins were observed in homogenates from inferior and lateral LV walls but neither from interventricular septum nor from right ventricle. SERCA2 expression was similar in all LV homogenates. The SERCA1a gene expression dramatically decreased between days 3 and 15, resulting in no significant overexpression (<5%) of SERCA proteins on day 15.


Figure 3
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  		Fig. 3. Representative Western blot analysis of SERCA1a in homogenates of LV inferolateral wall from 3 Ad.CMV.SERCA1a and 3 Ad.CMV.lacZ animals on day 3, and 3 Ad.CMV.SERCA1a animals on day 15. Controls (left) include homogenates of extensor digitorum longus (EDL) and LV from saline rabbit. A total of 50 µg of each LV homogenate and 1 µg of EDL were loaded.

 
To specify and quantify the effects of SERCA gene overexpression on SR Ca2+ transport, we measured the ATP-dependent oxalate-facilitated Ca2+ uptake activity of LV SR-enriched microsomes. Figure 4 shows the time course of Ca2+ uptake by homogenates of lateral LV wall from the three experimental groups 3 days after saline or adenovirus infusion. At pCa of 6.25 (close to the Kd of the enzyme), Ca2+ uptake increased gradually with time and tended to reach a plateau at 10 min (Fig. 4). In LV lateral wall homogenates from Ad.CMV.SERCA1a rabbits, Ca2+ uptake increased faster and was markedly higher at 10 min than in controls. The mean initial rate of Ca2+ uptake was 36% higher in LV transfected with Ad.CMV.SERCA1a than in controls (30.29 ± 4.42 vs. 19.42 ± 2.22 and 20.13 ± 1.75 nmol Ca2+·mg protein–1·min–1, respectively, P < 0.01), whereas total Ca2+ uptake at 10 min was 31% higher (105.2 ± 13.7 vs. 73.0 ± 9.5 and 73.8 ± 7.0 nmol Ca2+/mg protein, respectively, P < 0.01). These results were consistent with a larger number of SR Ca2+ pumps in transfected myocytes than in controls. This increase in Ca2+ uptake was positively related to the level of SERCA proteins overexpression (r2 = 0.36; P = 0.017). Within the interventricular septum, Ca2+ uptake was not significantly faster in Ad.CMV.SERCA1a animals than in the control groups (23.5 ± 2.5 vs. 19.6 ± 3.0 nmol Ca2+/mg protein, P = 0.42). On day 15, the difference between Ad.CMV.SERCA1a and controls groups was not statistically significant.


Figure 4
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  		Fig. 4. Time courses of Ca2+ uptake by homogenates of lateral LV wall on day 3. Saline (bullet), Ad.CMV.lacZ ({blacktriangleup}), and Ad.CMV.SERCA1a ({blacksquare}) are shown. *P < 0.01 vs. saline and Ad.CMV.lacZ (n = 7 animals in each group). Prot, protein.

 
Relationship between Ca2+ uptake and functional parameters. Figure 5 shows strong positive relations between the maximal rate of Ca2+ uptake and LV dP/dtmax observed after 2.5 µg·kg–1·min–1 dobutamine infusion on day 3 (r2 = 0.63; P < 0.001). In basal conditions (without dobutamine), a poor relationship was evident (r2 = 0.09). In contrast, strong positive relationships were observed with TDI-derived parameters of the transfected LV walls in basal conditions, such as Slat and the systolic peak strain rate (r2 = 0.61 and 0.68, respectively). Strong relationships were also observed between levels of SERCA protein overexpression and hemodynamics (LV dP/dtmax) or TDI-derived parameters (Slat and systolic peak strain rate) on day 3 (r2 = 0.54, 0.51, and 0.61, respectively; P < 0.001).


Figure 5
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  		Fig. 5. Relation between the maximal rate of Ca2+ uptake on day 3 and dP/dtmax during 2.5 µg·kg–1·min–1 dobutamine infusion (A), peak systolic velocities of lateral LV wall (B), and systolic strain rate of posterolateral LV wall (C). All animals, euthanized on day 3, are included in this analysis. SR, sarcoplasmic reticulum.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study shows that 1) percutaneous catheter-based delivery of Ad.CMV.SERCA1a to rabbit myocardium is efficient and results in a strong regional overexpression of SERCA proteins and an increase in the rate of SR Ca2+ uptake; 2) this effect is associated with an increase in LV contractility and relaxation for more than 7 days; and 3) TDI is particularly appropriate for assessing and monitoring effects on regional LV function, allowing gene therapy follow-up.

Efficiency of gene delivery is one of the technical obstacles that must be overcome before gene therapy in human heart failure becomes a practical proposition. Whereas systemic vector delivery is inefficient, catheter-based delivery to the coronary vasculature transfects from 1% (33) to 50% of cardiac myocytes (26, 27, 30). Most of the proposed experimental approaches, such as the aortic clamping, are poorly relevant to human heart failure. We have previously achieved a substantial improvement in gene transfer efficiency by increasing vessel permeability both by using catheter-mediated and pharmacological intervention and by increasing the vector residence time inside the cardiac vasculature (24). Here we tested adenoviral-mediated SERCA1a gene transfer with the same delivery technique. We confirm the high efficiency and reproducibility of this procedure, which results in a strong increase in SERCA activity in cardiac myocytes and in an increase in myocardial mechanical properties. With the use of this procedure in rabbit, the therapeutic gene is mainly delivered to the inferior and lateral LV walls because of the specific anatomy of the left coronary artery in rabbit; indeed, the coronary catheterization is selective for the large and dominant circumflex artery.

Ca2+ handling is similar in rabbit and human myocardium: after Ca2+ release and contraction, the SR accounts for 70% of cytosolic Ca2+ pumping by SERCA pumps, whereas the Na+/Ca2+-exchanger accounts for the remaining 30% (1). This offers the opportunity to alter the Ca2+ cycling by the means of SERCA overexpression more profoundly in the nonfailing rabbit heart than in small rodent where the SR already accounts for 90% of Ca2+ pumping. Indeed, we observed a 36% rise in SR Ca2+ uptake in the inferior and lateral LV walls, showing that the expressed SERCA1a pumps, resulting in a 20% overexpression of total SERCA proteins, were functional within the SR. This is similar to the 30–70% increase in Ca2+ uptake that was observed in studies using adenovirus-mediated SERCA gene transfer to isolated rat (4, 5) and rabbit myocytes (32). Our group (4) has previously shown that the distribution pattern of exogenous SERCA1a within the SR is similar to that of the endogenous SERCA2a protein, in a study based on in vitro gene transfer to adult cardiac myocytes, and others (6, 27) have shown that adenoviral-mediated SERCA2 gene transfer to rats with heart failure normalizes SR Ca2+ pumping.

SERCA1a overexpression also induced a significant increase in LV muscle performance. Based on invasive hemodynamic monitoring with a micromanometer placed in the LV cavity, our study shows a minimal dP/dt increase in Ad.CMV.SERCA1a-transfected animals. This only moderate alteration in global LV function can be explained by inhomogenous SERCA1a overexpression throughout the LV myocardium. In contrast, low-dose dobutamine infusion and heart pacing revealed more marked changes in global LV function. These results strongly suggest that the overexpressed SERCA1a protein was functional and coupled to phospholamban, of which its physiological inhibitory effect on SERCA pump activity is reduced by dobutamine infusion. At higher dobutamine concentrations, maximal and minimal dP/dt were similarly increased in transfected and control animals, suggesting that our SERCA overexpression is not sufficient to increase maximal LV mechanical performance in normal animals, i.e., to obtain supraphysiological effects. Previous in vitro studies in isolated rabbit cardiac myocytes showed similar results (2). Limiting factors may include the maximal capacity for Ca2+ storage within the SR and characteristics of the myofibrillar apparatus. Interestingly, pacing-mediated heart rate increase was associated with an increase in LV maximal and minimal dP/dt in Ad.CMV.SERCA1a animals only. This observation could be explained by the ability of the SR to accelerate Ca2+ uptake during the increase in heart rate, further supporting the functional relevance of the overexpression of SERCA proteins in cardiac myocytes.

Echocardiography has been widely used to study cardiac function in experimental models but is usually restricted to parameters such as LV dimensions and the shortening fraction. Recently, tissue Doppler analysis was used to determine LV filling pressures (2) and LV systolic function (5, 28). At variance with invasive hemodynamic studies, TDI analysis can be used to assess global but also regional LV function (5, 7, 8, 28), which was of particular relevance to our model, in which gene transfer to the myocardium is localized to inferior and lateral walls. Although the conventional LV shortening fraction was similar in all the groups, TDI analysis showed an increase in the velocities of the mitral annulus and lateral and inferior LV walls in Ad.CMV.SERCA1a animals only, as well as in the strain rate of the inferior LV wall. Mitral annulus velocities during systole and diastole reflect global LV function in the longitudinal axis and may be very sensitive to subtle changes in global LV function (8). The strain rate is an index of regional LV muscle function and was measured specifically within the posterolateral wall, which was targeted by adenovirus infusion. The strain rate of the posterolateral wall was markedly increased in Ad.CMV.SERCA1a animals only. Such an alteration was accurately monitored during follow-up. The strain rate is not the ideal parameter of LV muscle function, being load-sensitive like most other hemodynamic parameters. However, the strain rate is particularly relevant when load is similar in experimental groups, as that was the case in our study as shown by similar LV end-diastolic pressure. Interestingly, TDI values were closely related to the maximal rate of Ca2+ uptake and maximal dP/dt obtained after dobutamine infusion. This strengthens the relevance of TDI parameters for assessing regional changes in mechanical LV activity. Furthermore, this procedure allows the noninvasive functional monitoring of regional therapies and the follow-up of the effects with time.

Recently, Teucher et al. (32) pointed out that too high SERCA overexpression can be deleterious. By comparing two adenoviruses concentrations and the resulting levels of SERCA1a expression in isolated rabbit myocytes, these authors observed that highest levels of SERCA overexpression that were associated with impaired contractility that could be explained by excessive Ca2+ buffering within the SR. The level of this deleterious overexpression was not specified. On the other hand, a transgenic model with level of SERCA overexpression as high as 200% exhibited strong enhancement in LV function (25). In our in vivo study, we injected a single dose of Ad.CMV.SERCA1a that only resulted in a 12–27% overexpression of SERCA proteins. Higher levels of in vivo SERCA overexpression should be examined for checking the hypothesis where too strong SERCA overexpression is deleterious. It should be noticed that SERCA expression decreases in heart failure, which reduces such a risk.

Finally, adenoviral-based gene transfer resulted in transient gene expression in our model as well as in a number of previous adenovirus-based studies (10, 19). Transgene lacking has been explained by short-acting viral promoters as well as systemic and local immunitary reaction (18). Less immunogenic adenoviruses or other vectors, such adeno-associated viruses, are required for testing effects of long-term SERCA overexpression.

In conclusion, we show that Ad.CMV.SERCA1a transfection, using a clinically relevant method of gene delivery, significantly modulated cellular Ca2+ handling in the myocardium, resulting in improved LV function. These changes in regional myocardial function were accurately monitored by the means of TDI rather than conventional echography or hemodynamics. Further studies are required to examine long-term modulation of Ca2+ uptake in models of chronic heart failure.


   ACKNOWLEDGMENTS
 
We thank David Young for restyling the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Logeart, Service de Cardiologie, Hôpital Lariboisière, 2 rue Ambroise Paré, 75010 Paris, France (e-mail: damien.logeart{at}lrb.ap-hop-paris.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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