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(Circulation. 1997;95:423-429.)
© 1997 American Heart Association, Inc.

Articles

Physiological Effects of Adenoviral Gene Transfer of Sarcoplasmic Reticulum Calcium ATPase in Isolated Rat Myocytes

Roger J. Hajjar, MD; Jing X. Kang, MD, PhD; Judith K. Gwathmey, VMD, PhD; Anthony Rosenzweig, MD

the Cardiovascular Research Center and Cardiac Unit (R.J.H., A.R.), Medical Services, and Department of Preventive Medicine (J.X.K.), Massachusetts General Hospital, the Cardiovascular Disease and Muscle Research Laboratories (J.K.G.), and Harvard Medical School, Boston, Mass.

Correspondence to Anthony Rosenzweig, MD, Cardiovascular Research Center, Massachusetts General Hospital East, 149 13th St, Charlestown, MA 02107.

	Abstract

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Background In myocardial cells, relaxation is governed primarily by the sarcoplasmic reticulum (SR) Ca²⁺-ATPase transporting enzyme, which regulates Ca²⁺ sequestration into the SR. Human and experimental cardiomyopathies are associated with reduced SR Ca²⁺-ATPase activity.

Methods and Results To modify intracellular calcium mobilization, we created a recombinant adenovirus designed to overexpress the cardiac SR Ca²⁺-ATPase (SERCA2a) under the control of the Rous sarcoma virus (RSV). In neonatal rat myocytes, Ad.RSV.SERCA2a increased the expression of SERCA2a in a concentration-dependent and time-dependent fashion. Enhancement of SR Ca²⁺-ATPase activity was even greater than increases in SERCA2a protein content in cells infected with Ad.RSV.SERCA2a for 48 hours at a multiplicity of infection (MOI) from 0.1 to 10.0 pfu/cell. Intracellular calcium transients measured in the neonatal cells infected with Ad.RSV.SERCA2a were characterized by an abbreviation of the relaxation phase, an increase in peak [Ca²⁺]_i release, and a decrease in resting [Ca²⁺]_i levels. Ad.RSV.SERCA2a also enhanced the contraction of the myocardial cells as detected by shortening measurements.

Conclusions We found that adenovirus-mediated gene transfer of SR Ca²⁺-ATPase can modify intracellular calcium handling and shortening in myocardial cells. Such vectors should be useful in examining the role of reduced SERCA2a activity in the pathophysiology of heart failure and in developing strategies for gene therapy.

Key Words: genes • calcium • sarcoplasmic reticulum • SERCA2a

	Introduction

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The SR plays a major role in the regulation of intracellular calcium in cardiac muscle during contraction and relaxation.^{1 2} Calcium entry during activation of the myocardium induces Ca²⁺ release from the SR, which in turn activates the myofilaments. During relaxation, Ca²⁺ is taken up into the SR by the SR Ca²⁺-ATPase pump and is extruded from the cytoplasm by the Na⁺-Ca²⁺ exchanger and the sarcolemmal Ca²⁺-ATPase. In mammalian myocardium, the SR ATPase pump is responsible for most of the Ca²⁺ removal during relaxation on a beat-to-beat basis.¹ Changes in SR Ca²⁺ uptake function significantly affect cardiac excitation-contraction coupling. Relaxation abnormalities related to deficient SR Ca²⁺ uptake have been identified in human heart failure^{3 4 5 6 7 8} and in animal models of heart failure^{9 10 11 12 13 14 15 16 17 18} and have been associated with a reduction in SR Ca²⁺-ATPase activity.^{3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18} Steady-state SERCA2a mRNA levels are reduced in both failing human hearts and experimental models of heart failure.^{3 4 5 6 7 8 13} However, the results regarding SERCA2a protein levels in human heart failure have been more controversial. A number of investigators have shown reduced protein levels of SERCA2a in failing human hearts compared with nonfailing hearts,^{3 13} whereas other studies have not documented a significant difference in protein levels despite reduced SR Ca²⁺-ATPase activity.^{8 9} In cardiocytes, SR Ca²⁺-ATPase activity is tightly regulated not only by calcium levels but also by the interaction of SERCA2a with other proteins, such as phospholamban. The potential effects of increased SERCA2a protein on SR Ca²⁺-ATPase activity are therefore not straightforward. For this reason, examining the relationship between overexpression of SERCA2a in ventricular myocytes and SR Ca²⁺-ATPase activity may provide new insights into the regulation of SERCA2a.

Recently, replication-deficient recombinant adenoviral vectors have been used for gene transfer into myocardium and isolated cardiac myocytes.^{19 20 21 22 23 24 25} Adenovirus vectors have a number of important characteristics that make them well suited for myocardial gene transfer. These vectors effectively transfer exogenous reporter genes, such as ß-galactosidase and CAT, into rat myocardium by direct injection into the ventricular walls or by injection into the coronary arteries.^{19 20 21} In addition, recent reports demonstrate efficient gene transfer in vitro into isolated neonatal and adult rat ventricular myocytes.^{22 23 24} The efficiency and expression levels of adenovirus-mediated gene transfer in vivo and in vitro are substantially better than seen with other types of gene delivery such as direct plasmid DNA injection.^{19 20 21 22 23 24}

The goals of this study were (1) to overexpress SERCA2a in cultured neonatal myocytes and (2) to evaluate the biological effects of SERCA2a overexpression on intracellular calcium handling, myocyte shortening, and SR Ca²⁺-ATPase activity.

	Methods

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Construction of E1-Deleted Recombinant Adenoviral Vector Carrying SERCA2a cDNA
The rabbit SERCA2a cDNA was provided by Professor David H. MacLennan of the University of Toronto.^{26 27} The SERCA2a cDNA was subcloned between Not I and Xho I sites of the bacterial plasmid vector pAd.RSV4 (provided by Dr David A. Dichek, NIH), which uses the RSV long-terminal repeat as a promoter and the SV40 polyadenylation signal and contains Ad sequences 0 to 1 and 9 to 16 map units. The position and orientation of the SERCA2a cDNA were confirmed by restriction enzyme digestion and by polymerase chain reaction. The plasmid vector containing SERCA2a (pAd.RSV4.SERCA2a) was then cotransfected into 293 cells with pJM17 (provided by Dr Frank L. Graham, McMaster University). The homologous recombinants between the pAd.RSV.SERCA2a and pJM17 contain the SERCA2a cDNA substituted for E1. With this strategy, independent plaques were isolated and expression of SERCA2a protein was verified by immunostaining. A positive plaque was further plaque-purified, and protein expression was reconfirmed to yield the recombinant adenovirus, Ad.RSV.SERCA2a. This adenovirus is structurally similar to Ad.RSV.ßgal (kindly provided by Dr David Dichek), which carries a nuclear-targeted form of ß-galactosidase.²⁸ Ad.RSV.SERCA2a was then prepared as a high-titer stock by propagation in 293 cells as previously described.²⁵ Viral titer was determined to be 10¹⁰ pfu/mL by plaque assay as previously described.²⁵

Preparation of Neonatal Myocytes
Spontaneously beating cardiomyocytes were prepared from 1- to 2-day-old rats and cultured in F-10 medium (GIBCO BRL) in the presence of 5% FCS and 10% horse serum for 3 days as described previously.^{29 30} Measurements of cell shortening and cytosolic calcium were performed on neonatal myocytes cultured on round, coated glass coverslips (0.1 mm thick, 31 mm in diameter) in 35-mm culture dishes. Cells were counted with a hemocytometer. Approximately 5x10⁵ cells were plated in each coverslip.

Adenoviral Infection of Isolated Cells
Four days after isolation, the efficiency of adenoviral gene transfer was evaluated in the cultured rat myocytes by use of Ad.RSV.ßgal. Ad.RSV.ßgal was added in F-10 medium with 2% FCS at an MOI of 0.1, 1.0, and 10 pfu/cell. Cells were incubated at 37°C for 2 hours, then F-10 medium with 15% FCS added. After 24, 48, and 72 hours, cells were fixed with 2% glutaraldehyde, and ß-galactosidase expression was assayed by histochemical staining.³¹ The efficiency of gene transfer was significant starting at a virus concentration of 0.1 pfu/cell, with 100% of the cells being infected at 1 pfu/cell. In a similar manner, myocardial cells were infected with three concentrations of Ad.RSV.SERCA2a: 0.1, 1.0, and 10 pfu/cell for 48 hours. Infection with either Ad.RSV.ßgal or Ad.RSV.SERCA2a did not change the morphology of the cells.

Intracellular Calcium Measurements and Cell Shortening Detection
Measurements of intracellular calcium and cell shortening were performed as described earlier.^{29 30} Myocardial cells were loaded with the Ca²⁺ indicator fura 2 by incubation in medium containing 2 µmol/L fura 2-AM (Molecular Probes) for 30 minutes. The cells were then washed with PBS and allowed to equilibrate for 10 minutes in a light-sealed, temperature-controlled chamber (32°C) mounted on a Zeiss Axiovert 10 inverted microscope. The coverslip was superfused with a HEPES-buffered solution at a rate of 20 mL/h. A dual excitation spectrofluorometer was used to record fluorescence emissions (505 nm) elicited from exciting wavelengths at 360 and 380 nm. Intracellular calcium concentration was calculated according to the formula [Ca²⁺]_i=K_d(R-R_min)/(R_max-R)B, where R is the ratio of fluorescence of the cell at 360 and 380 nm; R_max and R_min represent the ratios of fura 2 fluorescence in the presence of saturating amounts of calcium and effectively "zero calcium," respectively; K_d is the dissociation constant of Ca²⁺ from fura 2; and B is the ratio of fluorescence of fura 2 at 380 nm in zero Ca²⁺ and saturating amounts of Ca²⁺. The myocardial cells were imaged with a CCD video camera (Javelin Electronics) attached to the microscope, and motion along a selected raster line segment was quantified by a video motion detector system (Ionoptix).

Preparation of SR Membranes From Isolated Rat Myocytes
To isolate SR membrane from cultured myocytes, we used a modified procedure from Harigaya and Schwartz³² and Wientzek and Katz.³³ Isolated neonatal myocytes were suspended in a buffer containing (in mmol/L) sucrose 300, PMSF 1, and PIPES 20, at pH 7.4. The myocytes were then disrupted with a homogenizer. The homogenates were centrifuged at 500g for 20 minutes. The resultant supernatant was centrifuged at 25 000g for 60 minutes to pellet the SR membrane. The pellet was resuspended in a buffer containing (in mmol/L) KCl 600, sucrose 30, and PIPES 20, frozen in liquid nitrogen, and stored at -70°C. Protein concentration was determined in these preparations by a modified Bradford procedure³⁴ using bovine serum albumin for the standard curve (Biorad).

Western Blot Analysis of SERCA2a and Phospholamban in SR Preparations
SDS-PAGE was performed on the isolated membranes from cell cultures under reducing conditions on a 7.5% separation gel with a 4% stacking gel in a Miniprotean II cell (Biorad). Proteins were then transferred to a Hybond-ECL nitrocellulose for 2 hours. The blots were blocked in 5% nonfat milk in Tris-buffered saline for 3 hours at room temperature. For immunoreaction, the blot was incubated with monoclonal anti-SERCA2a antibody diluted 1:2500 (Affinity BioReagents) or anti–cardiac phospholamban monoclonal IgG diluted 1:2500 (UBI) for 90 minutes at room temperature. After washing, the blots were incubated in a solution containing peroxidase-labeled goat anti-mouse IgG (dilution 1:1000) for 90 minutes at room temperature. The blot was then incubated in a chemiluminescence system and exposed to an X-Omat AR x-ray film (Fuji Films) for 1 minute. The densities of the bands were evaluated with NIH Image. Normalization was performed by dividing densitometric units of each membrane preparation by the protein amounts in each of these preparations. Serial dilution of the membrane preparations revealed a linear relationship between amounts of protein and the densities of the SERCA2a immunoreactive bands (data not shown).

SR Ca²⁺-ATPase Activity
SR Ca²⁺-ATPase activity assays were carried out according to Chu et al³⁵ based on pyruvate/NADH coupled reactions. With a photometer (Beckman DU 640) adjusted at a wavelength of 340 nm, oxidation of NADH (which is coupled to the SR Ca²⁺-ATPase) was assessed at 37°C in the homogenates by the difference of the total absorbance and basal absorbance. The reaction was carried out in a volume of 1 mL. All experiments were carried out in triplicate. The activity of the Ca²⁺-ATPase was calculated as absorbance/6.22xproteinxtime in nmol ATP·mg protein^-1·min^-1.

Statistical Analyses
Data were represented as mean±SEM for continuous variables. Student's t test was used to compare the means of normally distributed continuous variables. Parametric one-way ANOVA techniques were used to compare normally distributed continuous variables among uninfected groups of cells, Ad.RSV.ßgal-infected cells, and Ad.RSV.SERCA2a-infected cells.

	Results

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SERCA2a Expression in Myocytes Infected With Ad.RSV.SERCA2a
To determine whether SERCA2a is overexpressed in myocytes exposed to Ad.RSV.SERCA2a for 48 hours, we isolated membrane proteins from three groups of myocytes: (1) uninfected controls; (2) infected for 48 hours with Ad.RSV.ßgal at MOIs of 0.1, 1, and 10 pfu/cell; and (3) infected for 48 hours with Ad.RSV.SERCA2a at MOIs of 0.1, 1, and 10 pfu/cell, and Western blot analyses of SERCA2a and phospholamban were performed. As shown in Fig 1a, there was a dose-dependent increase in the amount of immunoreactive SERCA2a protein in myocardial cells infected with Ad.RSV.SERCA2a compared with uninfected cells or cells infected with Ad.RSV.ßgal at the same MOI. Levels of phospholamban did not differ among the three groups at the MOIs examined. SERCA2a protein levels normalized to total membrane protein increased with increasing MOI of virus from 0.1 to 10 pfu/cell (Fig 1b).

View larger version (143K): [in this window] [in a new window]   Figure 1. a, Immunoblots of SERCA2a and high-molecular-weight form of phospholamban (pentameric) in membrane preparations from uninfected myocytes and myocytes infected either with Ad.RSV.ßgal or Ad.RSV.SERCA2a at MOIs of 0.1, 1, and 10 pfu/cell. b, Protein levels of SR Ca2+-ATPase in noninfected myocytes (n=8), myocytes infected for 48 hours with 0.1, 1, and 10 pfu/cell of Ad.RSV.ßgal (n=7), and myocytes infected for 48 hours with 0.1, 1, and 10 pfu/cell of Ad.RSV.SERCA2a (n=9). *P<.05 compared with Ad.RSV.ßgal at MOI of 0.1 pfu/cell; #P<.05 compared with Ad.RSV.ßgal at MOI of 1 pfu/cell; P<.05 compared with Ad.RSV.ßgal at MOI of 10 pfu/cell. c, Immunoblots of SR Ca2+-ATPase after 24, 48, and 72 hours of infection with Ad.RSV.SERCA2a at MOI of 10 pfu/cell in neonatal myocytes. d, Protein levels of SR Ca2+-ATPase in noninfected myocytes (n=8) and myocytes infected with 10 pfu/cell of Ad.RSV.SERCA2a for 24 (n=4), 48 (n=9), and 72 hours (n=4).

To examine the time dependence of SERCA2a expression in the myocytes, Western blots were performed at 24, 48, and 72 hours after infection with 10 pfu/cell of Ad.RSV.SERCA2a. As shown by Fig 1c and 1d, protein expression increased with time and peaked at 48 hours after infection.

SR ATPase Activity in Myocytes Infected With Ad.RSV.SERCA2a
Maximal SR Ca²⁺-ATPase activity (Ca²⁺ 10 µmol/L) was measured by an optical assay in SR membrane homogenates from the isolated rat myocytes that were (1) uninfected, (2) infected with Ad.RSV.ßgal (0.1, 1.0, and 10 pfu/cell), or (3) infected with Ad.RSV.SERCA2a (0.1, 1.0, and 10 pfu/cell). The Ca²⁺-ATPase activities in the uninfected cell preparations and in the cells infected with Ad.RSV.ßgal (MOI, 10 pfu/cell) were 188±33 nmol·mg^-1·min^-1 (n=6) and 229±39 nmol·mg^-1·min^-1 (n=8), respectively. In the myocytes treated with Ad.RSV.SERCA2a, the Ca²⁺-ATPase activity was increased to 521±59 nmol·mg^-1·min^-1 (n=8). These results indicate that overexpression of SERCA2a results in an increase in the Ca²⁺-ATPase activity. To assess the changes in SR Ca²⁺-ATPase activity relative to changes in SERCA2a protein levels, we plotted the percent change in both ATPase activity and protein levels in myocytes infected with Ad.RSV.ßgal and Ad.RSV.SERCA2a at different MOIs. As shown in Fig 2, there was a dose-dependent increase in both SR Ca²⁺-ATPase protein and activity in myocytes infected with Ad.RSV.SERCA2a. However, for a given MOI, the increase in ATPase activity was greater than the increase in protein levels.

View larger version (11K): [in this window] [in a new window]   Figure 2. Percent change in SR Ca2+-ATPase activity and protein levels in myocyte preparations infected with Ad.RSV.ßgal and Ad.RSV.SERCA2a at different MOIs.

Characterization of Ca²⁺ Transients and Shortening in Rat Myocytes Infected With Ad.RSV.SERCA2a
To examine the effect of SERCA2a overexpression in rat myocytes, we measured intracellular calcium and shortening in uninfected controls and myocytes infected either with Ad.RSV.ßgal or with Ad.RSV.SERCA2a at an MOI of 10 pfu/cell. The cells were then loaded with fura 2, and the fluorescence and myocyte shortening were measured. As shown in Fig 3, myocytes infected with Ad.RSV.SERCA2a for 48 hours exhibited faster relaxation of both the calcium transient and shortening compared with uninfected myocytes or myocytes infected with Ad.RSV.ßgal. As shown in the Table, the peak of the calcium transient was increased in the Ad.RSV.SERCA2a group, the time to 80% relaxation of the calcium transient was reduced, and resting calcium levels were decreased. The extent of shortening was also increased in myocytes infected with Ad.RSV.SERCA2a compared with uninfected myocytes and myocytes infected with Ad.RSV.ßgal (Table).

View larger version (19K): [in this window] [in a new window]   Figure 3. Normalized [Ca2+]i transients and shortening measurements in a noninfected myocyte, a myocyte infected for 48 hours with 10 pfu/cell of Ad.RSV.ßgal, and a myocyte infected for 48 hours with 10 pfu/cell of Ad.RSV.SERCA2a.

View this table: [in this window] [in a new window]   Table 1. Effects of Ad.RSV.ßgal and Ad.RSV.SERCA2a on [Ca2+]i and Shortening in Isolated Myocytes

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we demonstrate the effective expression of SERCA2a in neonatal cardiac myocytes by adenoviral gene transfer. In addition, we show that protein overexpression results in a disproportionate increase in SR Ca²⁺-ATPase activity and in an enhancement of intracellular calcium uptake during relaxation.

Adenoviral Gene Transfer in Myocytes
Infection of isolated cells in vitro did not alter intracellular calcium handling of myocytes, as evidenced by the lack of effect on the intracellular calcium transient and shortening of Ad.RSV.ßgal-infected cardiocytes. Adenoviral gene transfer of SERCA2a was both dose-dependent and time-dependent. This dose response is similar to the one observed by Kass-Eisler et al ²² and Kirshenbaum et al,²³ who found a concentration-dependent increase in CAT activity with increasing concentrations of adenovirus (0.01 to 10 pfu/cell) carrying the CAT gene in neonatal myocytes and an increase in ß-galactosidase activity with increasing concentrations of adenovirus (1 to 1000 pfu/cell) containing a cytomegalovirus-driven ß-galactosidase gene in adult myocytes. In our studies, SERCA2a expression also increased in a time-dependent manner. At 24 hours, there was a 44% increase in SERCA2a expression and an 75% increase at 48 hours and at 72 hours at an MOI of 10 pfu/cell.

Overexpression of SR Ca²⁺-ATPase
The SR Ca²⁺-ATPase plays a key role in excitation-contraction coupling, transporting 2 mol Ca²⁺ into the lumen of the SR by hydrolyzing 1 mol of ATP.¹ It is responsible for lowering calcium during relaxation in myocytes and "loading" the SR with Ca²⁺ for the subsequent release and contractile activation.

Neonatal rat myocardium has a well-developed SR system, and Ca²⁺ homeostasis is dependent on Ca²⁺-induced Ca²⁺ release from the SR.³⁶ In our study, we found that Ad.RSV.SERCA2a infection increases the amount of SERCA2a in membrane preparations. However, the relationship between SERCA2a protein levels and biological activity is complex and tightly regulated in cardiocytes.^{37 38} The calcium pumping activity of this enzyme is strongly influenced by phospholamban, which is an integral part of the SR.¹ In the dephosphorylated state, phospholamban inhibits the Ca²⁺-ATPase, whereas phosphorylation of phospholamban by cAMP-dependent protein kinase and by Ca²⁺-calmodulin–dependent protein kinase reverses this inhibition.¹ Agents that increase cAMP levels in myocytes or activate calmodulin protein kinases enhance SR Ca²⁺-ATPase activity. Before this study, it was not clear to what extent overexpression of SERCA2a would increase Ca²⁺-ATPase activity. Thyroid hormone has been shown to increase the expression of SERCA2a and the Ca²⁺-ATPase activity in rat myocytes.^{39 40} However, thyroid hormone also decreases the expression of phospholamban. Therefore, increases in SR Ca²⁺-ATPase activity with thyroid hormone treatment cannot be attributed solely to SERCA2a overexpression. We found that increases in SERCA2a protein resulted in disproportionate increases in Ca²⁺-ATPase activity. This may reflect an increase in the amount of SERCA2a relative to phospholamban, resulting in decrease of the inhibitory effects by phospholamban. Although the SERCA2a overexpressed in these studies is from a rabbit clone, there are no large differences in Ca²⁺-ATPase activities or uptakes between rabbit and rat ventricular myocardium.^{32 34} Therefore, this species difference is unlikely to explain the disproportionate effect of overexpression on ATPase activity.

Examining the physiological effects of specifically increasing the SR Ca²⁺-ATPase activity has not been possible until now because increasing intracellular cAMP or calmodulin protein kinase affects a variety of channel activities.¹ Adenoviral gene transfer of SR Ca²⁺-ATPase provides an attractive system for further elucidation of the effects of SR Ca²⁺-ATPase on intracellular calcium handling. An enhancement in SR Ca²⁺ uptake rates is expected to lead to increased Ca²⁺ sequestered by the SR, which is then available for release, resulting in higher activation levels. We observed a shorter calcium transient and a lower resting [Ca²⁺], reflecting an enhanced Ca²⁺ uptake, as well as an increase in peak calcium levels, reflecting more Ca²⁺ available for release. These results support the concept that the SR Ca²⁺-ATPase is important both during relaxation in controlling the rate and amount of Ca²⁺ sequestered and during contraction in releasing the Ca²⁺ that is taken up by the SR. In addition, overexpression of SERCA2a resulted in enhanced shortening and faster relaxation. An increase in Ca²⁺ release would result in a larger amount of Ca²⁺ available for myofibrillar activation. Since contractile relaxation depends both on release of Ca²⁺ from the myofilaments and its uptake by the SR, our results showing a faster relaxation suggest that Ca²⁺ uptake by the SR is rate limiting during contractile relaxation.

The most likely explanation for the decrease in the time course of the calcium transient paralleling the increased protein expression and ATPase activities in the presence of Ad.RSV.SERCA2a is that the exogenous SERCA2a expressed in the neonatal myocytes is functional. An alternative possibility for the observed effect of Ad.RSV.SERCA2a on intracellular calcium handling is that the exogenous SERCA2a is not functional and is only competing for phospholamban, thereby relieving the inhibition on the endogenous SR Ca²⁺-ATPase pump. However, our experimental results of the maximal SR Ca²⁺-ATPase activity in the presence of Ad.RSV.SERCA2a do not support this hypothesis. Inhibition of phospholamban shifts the Ca²⁺-ATPase versus Ca²⁺ to the left (ie, higher Ca²⁺-ATPase for a given Ca²⁺) but does not affect the maximal Ca²⁺-ATPase activity. If exogenous SERCA2a were not functional and only competed for phospholamban, then the maximal SR Ca²⁺-ATPase activity measured should not have changed in the SR membranes isolated from myocytes infected by Ad.RSV.SERCA2a. This was shown very elegantly by Luo et al⁴¹ in the phospholamban-deficient mice. In our case, the Ca²⁺-ATPase activity was increased threefold at saturating Ca²⁺ (Fig 2). Therefore, our results suggest that the exogenous SERCA2a expressed in the neonatal myocytes incorporated into the SR did indeed function to transport Ca²⁺ across the SR membrane.

Conclusions
A number of disease states affect the expression of SR Ca²⁺-ATPase. Hyperthyroidism has been shown to increase the expression of SR Ca²⁺-ATPase and is associated with a shortening of the Ca²⁺ transient in working myocytes, whereas heart failure in both experimental models and humans has been associated with reduced SR Ca²⁺-ATPase activity and prolonged Ca²⁺ transient. We have shown that a recombinant adenovirus encoding the SR Ca²⁺-ATPase can effectively transfer this gene to isolated myocytes and modify the calcium transients as well as contractile responses. Although SR Ca²⁺-ATPase activity is tightly regulated, increased protein levels resulted in an even greater increase in activity. Whether a similar biological effect would be seen in myopathic or adult myocytes remains to be seen. Adenoviral gene transfer of SERCA2a should allow us to test its role in the pathogenesis of heart failure and cardiomyopathy.

	Selected Abbreviations and Acronyms

 

CAT = chloramphenicol acetyltransferase MOI = multiplicity of infection pfu = plaque-forming units RSV = Rous sarcoma virus SERCA2a = sarcoplasmic reticulum Ca2+-ATPase SR = sarcoplasmic reticulum

	Acknowledgments

 
This work was supported in part by grants from the NIH, K08-HL-03561 to Dr Hajjar, R29-HL-54202 to Dr Rosenzweig, and R01-HL-49574 and R44-HL-52249 to Dr Gwathmey; and grants to Dr Rosenzweig from Bristol Myers Squibb, the D.Y. and Joan Fu Fund for Cardiovascular Research, and the Merrill Lynch Research Fund. Dr Hajjar is Paul Dudley White Fellow of the American Heart Association, Massachusetts Affiliate. The authors would like to thank Prof MacLennan for kindly providing the SERCA2a clone to us, Dr Dichek for providing pAd.RSV4 and Ad.RSV.ßgal, and Dr Graham for providing the pJM17. The authors would also like to thank Drs Takashi Matsui, Ulrich Schmidt, and Ling Li for their assistance in Western blot analysis.

Received July 8, 1996; revision received September 4, 1996; accepted September 9, 1996.

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