Adenoviral Gene Transfer of Phospholamban in Isolated Rat Cardiomyocytes
Rescue Effects by Concomitant Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase
Abstract Phospholamban forms an integral part of the cardiac sarcoplasmic reticulum (SR) and regulates the activity of SR Ca2+-ATPase (SERCA2a). A number of studies have suggested a decrease in SERCA2a relative to phospholamban in heart failure. To test the hypothesis that changes in the relative abundance of phospholamban to SERCA2a could account for the pathophysiological abnormalities in Ca2+ handling observed in failing myocardium, we created a recombinant adenovirus designed to overexpress phospholamban (Ad.RSV.PL). In neonatal rat cardiomyocytes, Ad.RSV.PL increased the expression of phospholamban in a concentration-dependent fashion, reaching 280±43% at a multiplicity of infection (MOI) of 10.0 plaque forming units (pfu)/cell at 48 hours. The relationship between Ca2+-ATPase activity and [Ca2+] was shifted rightward in membrane preparations from cardiomyocytes infected with Ad.RSV.PL. Intracellular Ca2+ transients measured in the neonatal cells infected with Ad.RSV.PL (MOI, 10 pfu/cell) were characterized by (1) a significant prolongation of the relaxation phase (344±26 versus 710±56 milliseconds, P<.01), (2) a decrease in peak [Ca2+]i (967±43 versus 630±33 nmol/L, P<.01), and (3) an elevation in resting [Ca2+]i (143±14 versus 213±17 nmol/L, P<.05). Similarly, the time course of shortening was prolonged in myocytes infected with Ad.RSV.PL. These effects were partially restored by simultaneous transduction with an adeno-virus carrying SERCA2a. Cardiomyocytes infected with Ad.RSV.PL had an abnormal frequency response: a decrease in peak [Ca2+]i and an increase in resting [Ca2+]i with increasing frequency. These findings indicate that adenovirus-mediated gene transfer of phospholamban modifies intracellular Ca2+ handling and the frequency response in cardiomyocytes. Our results suggest that alterations in the ratio of phospholamban to SERCA2a could account for the abnormalities in Ca2+ handling observed in heart failure and that overexpression of SERCA2a can largely correct these abnormalities.
Phospholamban is an integral protein in the SR of mammalian cardiomyocytes involved in the regulation of Ca2+-ATPase, which transports Ca2+ into the SR.1 2 3 4 In the unphosphorylated state, phospholamban inhibits the SR Ca2+-ATPase by reducing its affinity for Ca2+.2 3 4 Phosphorylation of phospholamban at either the serine 16 site by cAMP-dependent protein kinase or the threonine 17 site by calmodulin-dependent mechanisms removes the inhibition to the SR Ca2+-ATPase.2 3 4 Phospholamban has been shown to be phosphorylated in situ and to contribute significantly to the positive inotropic response and the relaxant effects of β-agonism in the working heart.5 Recently, in elegant studies in phospholamban-deficient mice and in mice overexpressing phospholamban, Kranias and colleagues6 7 8 9 10 have shown that phospholamban tightly regulates SR Ca2+ transport and modulates the contractile response to β-agonism. However, during development, mice may alter their expression of other genes controlling Ca2+ kinetics and either mask or dilute the effects of transgene overexpression. Somatic gene transfer offers the advantage of direct overexpression of a specific gene without the confounding effects of developmental adaptations that may be present in transgenic animals. In addition, somatic gene transfer holds the potential for simultaneous independent overexpression of key proteins or for use in conjunction with genetically engineered animal models. Recently, replication-deficient recombinant adenovirus vectors have been used for gene transfer into myocardium both in vivo and in vitro.11 12 13 14 15 16 17 In neonatal rat cardiomyocytes, we recently demonstrated that a replication-deficient adenovirus overexpressing SERCA2a (Ad.RSV.SERCA2a) increased the expression of SERCA2a and SR Ca2+-ATPase activity in a concentration- and time-dependent fashion, abbreviated the intracellular Ca2+ transients, and enhanced the SR Ca2+-ATPase activity.17
A decrease in SR Ca2+-ATPase activity has been identified in a number of animal models of heart disease and in human heart failure associated with altered Ca2+ kinetics.18 19 20 21 22 23 24 25 26 However, there is controversy in the literature regarding the expression of SERCA2a and phospholamban in failing human hearts. Nevertheless, alterations in the relative ratio of phospholamban to SERCA2a seem to be an important characteristic of both experimental and human heart failure.24 25 26 Adenoviral gene transfer of phospholamban allowed us to test whether increasing phospholamban relative to SERCA2a could account for the abnormalities of Ca2+ handling observed in myopathic hearts and to also test the ability of SERCA2a to rescue this phenotype.
Materials and Methods
Construction of E1-Deleted Recombinant Adenovirus Vectors
The construction of Ad.RSV.SERCA2a has been described in detail previously.17 Ad.RSV.βgal, which carries a nuclear localizing form of β-galactosidase, was kindly provided by Dr David Dichek (Gladstone Institute for Cardiovascular Diseases, San Francisco, Calif).27 The rabbit phospholamban cDNA was provided by Prof David H. MacLennan from the University of Toronto (Canada).28 The phospholamban cDNA was subcloned into the bacterial plasmid vector pAdRSV4 (provided by Dr Beverly Davidson, University of Iowa, Iowa City), which uses the RSV long terminal repeat as a promoter and the SV40 polyadenylation signal and contains map units with adenovirus sequences from 0 to 1 and from 9 to 16. The position and orientation of the phospholamban cDNA were confirmed by restriction enzyme digestion and by polymerase chain reaction. The plasmid vector containing phospholamban (pAd.RSV.PL) was then cotransfected into 293 cells with pJM17 (provided by Dr Frank L. Graham, McMaster University, Hamilton, Canada). The homologous recombinants between pAd.RSV.PL and pJM17 contain the phospholamban cDNA substituted for E1. By use of this strategy, independent plaques were isolated, and expression of phospholamban protein was verified by immunostaining. A positive plaque was further plaque-purified, and protein expression was reconfirmed to yield the recombinant adenovirus Ad.RSV.PL. This adenovirus is structurally similar to Ad.RSV.βgal and to Ad.RSV.SERCA2a.17 The recombinant viruses were prepared as high-titer stocks by propagation in 293 cells as previously described.29 The titers of stocks used for these studies were as follows: 3.1×1010 pfu/mL for Ad.RSV.PL, 2.6×1010 pfu/mL for Ad.RSV.SERCA2a, and 2.7×1010 pfu/mL for Ad.RSV.βgal, with a particle-to-pfu ratio of 40:1, 42:1, and 37:1, respectively.
Preparation of Neonatal Cardiomyocytes
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% fetal calf serum and 10% horse serum for 3 days as described previously.30 31 Measurements of cell shortening and cytosolic Ca2+ were performed on neonatal cardiomyocytes cultured on round, coated, glass coverslips (0.1-mm thickness, 31-mm diameter) in 35-mm culture dishes. Cells were counted using a hemocytometer. Approximately 5×105 cells were plated in each coverslip.
Adenoviral Infection of Isolated Cells
Our previous work has demonstrated that the efficiency of gene transfer is significant starting at a virus concentration of 0.1 pfu/cell, with nearly 100% of the cells being infected at 1 pfu/cell. In three different infection experiments with increasing concentrations of Ad.RSV.βgal, the percentages of cells expressing βgal, after 48 hours, by histochemical staining in 10 different high-power fields were 98±2% (MOI, 1 pfu/cell), 99±1% (MOI, 10 pfu/cell), and 100% (MOI, 100 pfu/cell). In a similar manner, myocardial cells were infected with three concentrations of Ad.RSV.PL: 1.0, 10, and 100 pfu/cell for 48 hours. Infection with either Ad.RSV.βgal, Ad.RSV.PL, or Ad.RSV.SERCA2a did not change the morphology of the cells. For each infection experiment with the adenovirus, we used one myocyte to measure functional parameters.
Intracellular Ca2+ Measurements and Cell Shortening Detection
Measurements of intracellular Ca2+ and cell shortening were performed as described earlier.17 30 31 Myocardial cells were loaded with the Ca2+ indicator fura 2 by incubating the cells 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 (Zeiss). The coverslip was superfused with a HEPES-buffered solution at a rate of 20 mL/h. Cells were stimulated at different frequencies (0.1 to 2.0 Hz) using an external stimulator (Grass Instruments). A dual excitation spectrofluorometer (IONOPTIX) was used to record fluorescence emissions (505 nm) elicited from exciting wavelengths of 360 and 380 nm. [Ca2+]i was calculated according to the following formula: [Ca2+]i=Kd(R−Rmin)/(Rmax−R)B, where R is the ratio of fluorescence of the cell at 360 and 380 nm; Rmax and Rmin represent the ratios of fura 2 fluorescence in the presence of saturating amounts of Ca2+ and effectively “zero Ca2+,” respectively; Kd 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+. Unless otherwise stated, measurements of peak [Ca2+]i and resting [Ca2+]i were made when cells were stimulated at 1 Hz. Specifically, measurements of resting [Ca2+]i were made at the end of diastole. High-contrast microspheres attached to the cell surface of the cardiomyocytes were imaged using a charge-coupled device video camera attached to the microscope, and motion along a selected rastor line segment was quantified by a video motion detector system (IONOPTIX).
Preparation of SR Membranes From Isolated Rat Cardiomyocytes
To isolate SR membrane from cultured cardiomyocytes, we used a procedure modified from Harigaya and Schwartz32 as well as Wientzek and Katz.33 Isolated neonatal cardiomyocytes were suspended in a buffer containing (mmol/L) sucrose 300, phenylmethylsulfonyl fluoride 1, and PIPES 20, at pH 7.4. The cardiomyocytes 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-enriched membrane. The pellet was resuspended in a buffer containing (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 (Bio-Rad).
Western Blot Analysis of Phospholamban and SERCA2a 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 (Bio-Rad). Proteins were then transferred to 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 1:2500 diluted monoclonal anti-SERCA2 antibody (Affinity BioReagents) or 1:2500 diluted anti-cardiac phospholamban monoclonal IgG (UBI) for 90 minutes at room temperature. After a 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 x-ray film (Fuji Films) for 1 minute. The densities of the bands were evaluated using 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 on the basis of pyruvate/NADH-coupled reactions. By use of 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 membrane preparations 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 follows: Δabsorbance/6.22×protein×time (in nmol ATP/mg protein×min). The measurements were repeated at different [Ca2+] levels. The effect of the specific Ca2+-ATPase inhibitor CPA at a concentration range of 0.001 to 10 μmol/L was also studied in these preparations.36 37
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.βgal-infected cells, Ad.RSV.PL-infected cells, and Ad.RSV.SERCA2a-infected cells.
Phospholamban Expression in Cardiomyocytes Infected With Ad.RSV.PL and Ad.RSV.SERCA2a
We quantified the levels of phospholamban in membrane preparations from uninfected cardiomyocytes infected with increasing concentrations of Ad.RSV.PL (MOI, 1, 10, and 100 pfu/cell). As shown in Fig 1⇓, there was a 4-fold increase in phospholamban protein levels in a dose-dependent fashion. Over this range, there was a dose-dependent increase in the protein expression of phospholamban between 1 and 10 pfu/cell but no further increases between 10 and 100 pfu/cell. Coinfection of Ad.RSV.SERCA2a with Ad.RSV.PL produced an increase in protein expression of both SERCA2a and phospholamban, as shown by the immunoblot in Fig 2a⇓. As shown in Fig 2b⇓, there were no significant differences between the phospholamban protein levels in the group of myocytes infected with Ad.RSV.PL alone at an MOI of 10 pfu/cell and the group of myocytes infected with Ad.RSV.PL at an MOI of 10 pfu/cell and Ad.RSV.SERCA2a at an MOI of 10 pfu/cell (P>.2). Similarly, there were no significant differences between the SERCA2a protein levels in the group of myocytes infected with Ad.RSV.SERCA2a alone at an MOI of 10 pfu/cell and the group of myocytes infected with Ad.RSV.PL at an MOI of 10 pfu/cell and Ad.RSV.SERCA2a at an MOI of 10 pfu/cell (P>.2).
SR ATPase Activity in Cardiomyocytes Infected With Ad.RSV.Pl and Ad.RSV.SERCA2a
The effects of phospholamban overexpression on SR ATPase activity were measured in membranes from cardiomyocytes infected with Ad.RSV.PL (MOI, 10 pfu/cell). As shown in Fig 3a⇓, the relationship between ATPase activity and Ca2+ was shifted to the right in the preparations from cardiomyocytes overexpressing phospholamban compared with the uninfected preparations without changing maximal Ca2+-ATPase activity. Coinfection with Ad.RSV.SERCA2a restored the Ca2+-ATPase activity and also increased the maximal Ca2+-ATPase activity. To verify that the ATPase activity we measured from the membrane preparations was SR-related, we used the specific inhibitor CPA after maximally activating the SR Ca2+-ATPase with 10 μmol/L of Ca2+. As shown in Fig 3b⇓, CPA inhibited the SR Ca2+-ATPase activity in a dose-dependent fashion in all three membrane preparations (uninfected, Ad.RSV.PL, and Ad.RSV.PL+Ad.RSV.SERCA2a).
Characterization of Ca2+ Transients and Shortening in Rat Cardiomyocytes Infected With Ad.RSV.PL
To examine the effect of phospholamban overexpression on Ca2+ handling, we measured intracellular Ca2+ in uninfected cardiomyocytes and cardiomyocytes infected either with Ad.RSV.βgal or with Ad.RSV.PL (±Ad.RSV.SERCA2a). As shown in Fig 4a⇓, cardiomyocytes infected with Ad.RSV.βgal did not affect the Ca2+ transient or shortening compared with control uninfected cardiomyocytes. As depicted in Fig 4b⇓, the Ca2+ transient and shortening were significantly altered with increasing concentrations of Ad.RSV.PL (MOI, 1, 10, and 100 pfu/cell): observed changes included prolongation of the Ca2+ transient and shortening and a decrease in the peak Ca2+. These results, summarized in the Table⇓, show that there was a dose-dependent prolongation of the Ca2+ transient and mechanical shortening up to 10 pfu/cell, with no further significant prolongation at 100 pfu/cell. Similarly, peak [Ca2+] decreased up to 10 pfu/cell, with no further significant decrease at 100 pfu/cell. Coinfection with Ad.RSV.SERCA2a (MOI, 10 pfu/cell) restored both the Ca2+ transient and the shortening to near normal levels, as shown in Fig 4c⇓. Fig 5⇓ shows a significant decrease in mean peak [Ca2+], a significant increase in mean resting [Ca2+], and a significant prolongation of the Ca2+ transient in the group of cardiomyocytes infected with Ad.RSV.PL (MOI, 10 pfu/cell) compared with uninfected cells (panels a through c, respectively). These effects were partially restored by the addition of Ad.RSV.SERCA2a (MOI, 10 pfu/cell) (Fig 5⇓). Similarly, the time course of shortening was significantly prolonged in cardiomyocytes infected with Ad.RSV.PL at an MOI of 10 pfu/cell (time to 80% relaxation, from 387±22 to 780±44 milliseconds; P<.05; n=12), whereas coinfection with Ad.RSV.SERCA2a restored the time course to normal (405±25 milliseconds, n=10, P>.1 compared with uninfected cells).
Effects of Isoproterenol on Ca2+ Transients in Cardiomyocytes
To evaluate the role phospholamban plays in the lusitropic effect of β-agonism, we evaluated the effects of isoproterenol in cardiomyocytes overexpressing phospholamban. Increasing concentrations of isoproterenol resulted in a decrease in the relaxation time of the Ca2+ transient in uninfected cells (Fig 6⇓). At low doses of isoproterenol, cardiomyocytes infected with Ad.RSV.PL (MOI, 10 pfu/cell) demonstrated a larger decrease in the time to 80% relaxation. However, at maximal isoproterenol stimulation (1 μmol/L), there was no significant difference between the [Ca2+] time course of Ad.RSV.PL-infected and uninfected control cardiomyocytes.
Effects of Increasing the Stimulation Frequency in Cardiomyocytes
The positive contractile response to increasing frequency of stimulation is thought to be mediated by the SR and by the Ca2+ loading of the SR. We tested the effects of slowing the rate of Ca2+ uptake by overexpressing phospholamban on the frequency response of cardiomyocytes. We increased the stimulation frequency from 0.5 to 2 Hz in uninfected cardiomyocytes and in cardiomyocytes infected with Ad.RSV.PL (MOI, 10 pfu/cell). As shown in Fig 7⇓, cardiomyocytes infected with Ad.RSV.PL (MOI, 10 pfu/cell) exhibited a significant increase in resting Ca2+ not evident in uninfected cells. Furthermore, coinfection with Ad.RSV.SERCA2a (MOI, 10 pfu/cell) restored the frequency response to normal.
In the present study, we used adenoviral gene transfer to overexpress phospholamban alone and in combination with SERCA2a. Physiological effects of expression on intracellular Ca2+ handling and SR Ca2+-ATPase activity were evaluated. This approach allows us to study dose-dependent interactions of these two key SR proteins in working cardiomyocytes.
Adenoviral Gene Transfer in Cardiomyocytes
A number of investigators have shown that gene transfer using adenovirus is effective in mammalian myocardium both in vivo and in vitro.11 12 13 14 15 16 We had previously shown that adenoviral gene transfer of SERCA2a was both dose dependent and time dependent in rat neonatal cardiomyocytes.17 In the present study using an adenovirus encoding phospholamban under the same promoter, RSV, there was a 4-fold increase in phospholamban, which was also dose dependent. The smaller size of phospholamban compared with SERCA2a (6 kD in its monomer form compared with 110 kD) may explain, at least in part, the more effective protein expression by Ad.RSV.PL than by Ad.RSV.SERCA2a under similar conditions. Nevertheless, using these recombinant adenoviruses, we were able to achieve significant overexpression of phospholamban and SERCA2a, individually and in combination. Coinfection with both Ad.RSV.PL and Ad.RSV.SERCA2a mediated overexpression of both SERCA2a and phospholamban that was the same as the expression from infection with either Ad.RSV.PL or Ad.RSV.SERCA2a alone. To our knowledge, this is the first demonstration that coinfection with more than one recombinant adenovirus is feasible in myocardial cells. The ability to simultaneously manipulate expression of multiple proteins in the context of primary myocytes is an advantage of somatic gene transfer for the study of interacting components of complex systems.
Physiological Consequences of Altering the Phospholamban-to-SERCA2a Ratio
The expression of phospholamban relative to SERCA2a has been shown to be altered in a number of disease states.25 26 In hypothyroidism phospholamban levels are increased, whereas in hyperthyroidism phospholamban levels are decreased.38 39 An increased ratio of phospholamban to SERCA2a is an important characteristic of both human and experimental heart failure.18 19 20 21 22 23 24 25 26 Both experimental and human heart failure are characterized by a prolonged Ca2+ transient and impaired relaxation. In the present study, increasing levels of phospholamban relative to SERCA2a significantly altered intracellular Ca2+ handling in the isolated cardiomyocytes by prolonging the relaxation phase of the Ca2+ transient, decreasing Ca2+ release, and increasing resting Ca2+. These results support the hypothesis that altering the relative ratio of phospholamban to SERCA2a can account for the abnormalities in Ca2+ handling observed in failing ventricular myocardium. In addition, the present study shows that overexpressing SERCA2a can largely “rescue” the phenotype created by increasing the phospholamban-to-SERCA2a ratio. This rescue effect by SERCA2a is especially encouraging, since it suggests that restoring the normal phospholamban-to-SERCA2a ratio through somatic gene transfer could correct the abnormalities of Ca2+ handling and contraction seen in failing hearts.
Effect of Adenoviral Gene Transfer on SR Ca2+-ATPase
The SR Ca2+-ATPase plays a key role in excitation-contraction coupling, lowering Ca2+ during relaxation in cardiomyocytes, and “loading” the SR with Ca2+ for the subsequent release and contractile activation.1 The Ca2+-pumping activity of this enzyme is influenced by phospholamban. In the unphosphorylated 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 2 3 4 Therefore, an increase in phospholamban content should decrease the affinity of the SR Ca2+ pump for Ca2+. As shown in Fig 4⇑, overexpression of phospholamban shifted the relationship between SR Ca2+-ATPase activity and Ca2+ to the right, indicating a decrease of the sensitivity of the SR Ca2+ to pump to Ca2+. However, there was no change in the maximal Ca2+-ATPase activity in the Ad.RSV.PL-infected cardiomyocytes. Our findings are in agreement with the results of Odermatt et al,40 who found that the Vmax of the Ca2+-ATPase of cardiac SR is not altered by interaction with phospholamban and phosphorylation. These findings are also similar to those of Kadambi et al,9 who showed that in mice overexpressing phospholamban, the affinity of the SR Ca2+ pump for Ca2+ was decreased but that the maximal velocity of the SR Ca2+ uptake was not changed. From the present experiment, it can also be concluded that phospholamban affects the affinity of the SR Ca2+ pump for Ca2+ without changing the maximal ATPase activity. The concomitant overexpression of SERCA2a and phospholamban restored the ATPase activity and also increased the maximal Ca2+-ATPase activity. This brings further evidence that the expression of additional SR Ca2+-ATPase pumps can overcome the inhibitory effects of phospholamban.
Effects of Adenoviral Gene Transfer on Ca2+ Kinetics
Adenoviral gene transfer of phospholamban provides an attractive system for further elucidation of the effects of inhibiting SR Ca2+-ATPase on intracellular Ca2+ handling. A decrease in SR Ca2+ uptake rates is expected to lead to a smaller amount of Ca2+ sequestered by the SR, resulting in a smaller amount of Ca2+ release. In neonatal cardiomyocytes, we observed a significantly prolonged Ca2+ transient and a higher resting [Ca2+] reflecting the decreased Ca2+ uptake and a decrease in peak [Ca2+] levels reflecting less Ca2+ available for release. These results support the concept that the SR Ca2+-ATPase is important during relaxation by controlling the rate and amount of Ca2+ sequestered and during contraction by releasing the Ca2+ that is taken up by the SR. Overexpression of both phospholamban and SERCA2a partially restored the Ca2+ transient; however, the time course of the Ca2+ transient was still prolonged in cardiomyocytes infected with both Ad.RSV.SERCA2a and Ad.RSV.PL. This finding was somewhat surprising, since the SR Ca2+-ATPase activity was restored to normal and even enhanced in cardiomyocytes infected with both Ad.RSV.SERCA2a and Ad.RSV.PL.
Effects of Adenoviral Gene Transfer on the Response to β-Agonism
Phospholamban has been shown to play a key role in modulating the response of agents that increase cAMP levels in cardiomyocytes.5 Since phosphorylation of phospholamban reduces the inhibition to the SR Ca2+ pump, thereby enhancing the SR Ca2+-ATPase, we were specifically interested in evaluating the effects of β-agonism on the relaxation phase of the Ca2+ transient. In the basal state, the overexpression of phospholamban significantly prolongs the Ca2+ transient. As shown in Fig 6⇑, at maximal isoproterenol stimulation, the time course of the Ca2+ transients in the uninfected cardiomyocytes and the cardiomyocytes infected with Ad.RSV.PL were decreased to the same level. Our findings demonstrate that phospholamban plays a major role in the enhanced relaxation of the heart to β-agonism. In addition, it corroborates our findings that phospholamban decreases the affinity of the SR Ca2+ pump for Ca2+ but does not decrease the maximal Ca2+ uptake rate.
Effects of Adenoviral Gene Transfer on the Frequency Response
The response to increasing stimulation frequencies in mammalian cardiomyocytes is governed by the SR. A number of investigators have proposed that the decreased SR Ca2+-ATPase activity observed in myopathic hearts is responsible for the negative force-frequency relationship in myopathic ventricles.20 22 Therefore, we tested the hypothesis that inhibiting the SR Ca2+-ATPase by overexpressing phospholamban would alter the frequency response of cardiomyocytes. In the uninfected cardiomyocytes, an increase in stimulation frequency did not significantly alter either peak or resting [Ca2+]i. This response is typical of rat cardiomyocytes that have either a flat response to increasing frequency of stimulation or a decrease in contractile force.41 However, in cardiomyocytes infected with Ad.RSV.PL, there was a significantly greater increase in resting [Ca2+] and a decrease in peak [Ca2+]. These results would suggest that diminished SR Ca2+ uptake leads to a diminished Ca2+ release, which becomes even more accentuated at higher frequencies of stimulation.
In the present study, we have used recombinant adenovirus vectors to alter expression of two key SR proteins alone and in combination in isolated cardiomyocytes. We have characterized the biochemical and physiological effects of phospholamban overexpression and found that (1) the Ca2+-ATPase–[Ca2+ ] relationship was shifted rightward; (2) intracellular Ca2+ transients measured in the neonatal cells exhibited a significant prolongation of the relaxation phase, a decrease in peak [Ca2+]i, and an elevation in resting [Ca2+]; (3) an enhanced responsiveness to β-agonist stimulation; and (4) an abnormal response to increasing frequency stimulation. Our results support the hypothesis that alterations in the ratio of phospholamban to SERCA2a could account for the abnormalities in Ca2+ handling observed in heart failure and that overexpression of SERCA2a can largely correct these abnormalities.
Targeting phospholamban and SERCA2a by somatic gene transfer in animal models of heart failure in order to correct the alterations in the relative ratio of phospholamban to SERCA2a will allow us to test whether this strategy will improve contractile function in the failing heart.
Selected Abbreviations and Acronyms
|β-gal (combination form)||=||β-galactosidase|
|Ad (combination form)||=||adenovirus|
|MOI||=||multiplicity of infection|
|PL (combination form)||=||phospholamban|
|RSV||=||Rous sarcoma virus|
|SERCA2a||=||cardiac SR Ca2+-ATPase|
|SV40||=||simian virus 40|
This study was supported in part by grants from the National Institutes of Health (HL-50361 and HL-57623 to Dr Hajjar and HL-54202 and AI-40970 to Dr Rosenzweig), by the Merrill Research Fund for Cardiovascular Research, and by the D.Y. and Joan Fu fund for Cardiovascular Research.
This manuscript was sent to Ketty Schwartz, Associate Editor, for review by expert referees, editorial decision, and final disposition.
- Received January 31, 1997.
- Accepted May 29, 1997.
- © 1997 American Heart Association, Inc.
Barry WH, Bridge JHB. Intracellular calcium homeostasis in cardiac myocytes. Circulation. 1993;87:1806-1815.
Sasaki T, Inui M, Kimura Y, Kuzuya Y, Tada M. Molecular mechanism of regulation of Ca2+ pump ATPase by phospholamban in cardiac sarcoplasmic reticulum. J Biol Chem. 1992;25:1674-1679.
Toyofuku T, Kurzydlowski K, Tada M, MacLennan DH. Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca2+ ATPase of sarcoplasmic reticulum J Biol Chem. 1994;268:2809-2815.
Toyofuku T, Kurzydlowski K, Tada M, MacLennan MH. Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca2+-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban. J Biol Chem. 1994;37:22929-22932.
Karczewski P, Bartel S, Krause E-G. Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts. Biochem J. 1990;266:115-122.
Luo W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-agonist stimulation. Circ Res. 1994;75:401-409.
Luo W, Wolska BM, Grupp IL, Harrer JM, Haghighi K, Ferguson DG, Slack JP, Grupp G, Doetschman T, Solaro RJ, Kranias EG. Phospholamban gene dosage effects in the mammalian heart. Circ Res. 1996;78:839-847.
Hoit BD, Khoury SF, Kranias EG, Ball N, Walsh RA. In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency. Circ Res. 1995;77:632-637.
Koss KL, Kranias EG. Phospholamban: a prominent regulator of myocardial contractility. Circ Res. 1996;79:1059-1063.
French BA, Mazur W, Geske RS, Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation. 1994;90:2414-2424.
Barr E, Carroll J, Kalynych AM, Tripathy SK, Kozarsky K, Wilson JM, Leiden JM. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Hum Gene Ther. 1994;1:51-58.
Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res. 1993;73:1202-1207.
Kass-Eisler A, Falck-Pedersen E, Alvira M, Rivera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA. Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and vivo. Proc Natl Acad Sci U S A. 1993;90:11498-11502.
Kirshenbaum LA, MacLennan WR, Mazur W, French BA, Schneider MD. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J Clin Invest. 1993;92:381-387.
Johns DC, Nuss HB, Chiamvimonvat N, Ramza BM, Marban E, Lawrence JH. Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). J Clin Invest. 1995;95:1152-1158.
Hajjar RJ, Kang JX, Gwathmey JK, Rosenzweig A. Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum Ca2+ ATPase in isolated rat myocytes. Circulation. 1997;95:423-429.
Arai M., Matsui H, Periasamy M. Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res. 1994;74:555-564.
Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, Morgan JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res. 1987;61:70-76.
Gwathmey JK, Slawsky MT, Hajjar RJ, Briggs GM, Morgan JP. Role of intracellular calcium handling in force interval relationships of human ventricular myocardium. J Clin Invest. 1990;85:1599-1628.
Gwathmey JK, Liao R, Hajjar RJ. Intracellular free calcium in hypertrophy and failure. In: Lorell BH, Grossman WG, eds. Diastolic Relaxation in the Heart. Boston, Mass: Kluwer Academic Publishing; 1994:55-64.
Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium. Circ Res. 1994;75:434-442.
Schwinger RHG, Bohm MR, Schmidt U, Erdmann E. Unchanged sarcoplasmic reticulum Ca2+-ATPase in nonfailing and failing human myocardium. Circulation. 1995;86:1402-1410.
Meyer M, Schillinger W, Pieske B, Holubarsch C, Heilmann C, Posival H, Kuwajima G, Mikoshiba K, Just H, Hasenfuss G. Alterations of sarcoplasmic reticulum proteins in failing human dilated cardiomyopathy. Circulation. 1995;92:778-784.
Rockman HA, Ono S, Ross JR, Jones LR, Karimi M, Bhargava V, Ross RJ, Chien KR. Molecular and physiological alterations in murine ventricular dysfunction. Proc Natl Acad Sci U S A. 1994;91:2694-2698.
Kiss E, Ball NA, Kranias EG, Walsh RA. Differential changes in cardiac phospholamban and sarcoplasmic reticular Ca2+-ATPase protein levels: effects on Ca2+ transport and mechanics in compensated pressure-overload hypertrophy and congestive heart failure. Circ Res. 1995;77:759-764.
Dong G, Schulick A, DeYoung MB, Dichek DA. Identification of a cis-acting sequence in the human PAI-1 gene that mediates TGF-1 responsiveness in endothelium in vivo. J Biol Chem. 1996;271:29969-29977.
Lytton J, MacLennan DH. Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene. J Biol Chem. 1988;263:15024-15031.
Graham FL, Preyec L. Manipulation of adenovirus vectors In: Murray EJ, ed. Methods in Molecular Biology: Gene Transfer and Expression Protocols. Clinton, NJ: Humana Press; 1991:109-128.
Kang JX, Xiao Y-F, Leaf A. Free, long-chain, polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes. Proc Natl Acad Sci U S A. 1995;92:3097-4001.
Harigaya S, Schwartz A. Rate of calcium binding and uptake in normal animal and failing human cardiac muscle-membrane vesicles (relaxing system) and mitochondria. Circ Res. 1969;25:781-794.
Schwinger RHG, Bohm M, Schmidt U, Karczewski P, Bavndiek U, Flesch M, Krause E-G, Erdmann E. Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+ uptake and Ca2+ ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts. Circulation. 1995;92:3220-3228.
Baudet S, Shaoulian R, Bers DM. Effects of thapsigargin and cyclopiazonic acid on twitch force and sarcoplasmic reticulum Ca2+ content of rabbit ventricular muscle. Circ Res. 1993;73:813-819.
Kiss E, Jakab G, Kranias EG, Edes I. Thyroid hormone–induced alterations in phospholamban protein expression: regulatory effects on sarcoplasmic reticulum Ca2+ transport and myocardial relaxation. Circ Res. 1994;75:245-251.
Arai E, Otsu K, MacLennan DH, Alpert NR, Periasamy M. Effect of thyroid hormone on the expression of mRNA encoding sarcoplasmic reticulum proteins. Circ Res. 1991;69:266-276.
Odermatt A, Kurzydlowski K, MacLennan DH. The vmax of the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) is not altered by Ca2+/calmodulin-dependent phosphorylation or by interaction with phospholamban. J Biol Chem. 1996;271:14206-14213.
Orchard CH, Lakatta EG. Intracellular calcium transients and developed tensions in rat heart muscle: a mechanism for the negative interval-strength relationship. J Gen Physiol. 1995;86:637-651.