INTRODUCTION

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PHOSPHOLEMMAN (PLM), a 72-amino acid integral membrane phosphoprotein with a single transmembrane domain, is a major sarcolemmal substrate for protein kinases A and C in heart and skeletal muscle (14, 18, 20). Its physiological function is largely unknown. Early work based on overexpression of PLM in Xenopus oocytes suggested that PLM was a hyperpolarization-activated anion-selective channel (16). More recent studies, however, indicated that PLM interacts with endogenous oocyte anion channels because expression of nonchannel hydrophobic peptides induced similar currents in Xenopus oocytes (22, 25). When reconstituted in lipid bilayers, PLM formed a channel that was highly selective for taurine (2). In addition, it was recently recognized that PLM belongs to the FXYD family of small ion transport regulators (23). Thus current evidence suggests that PLM 1) can be a channel, a channel subunit, or an ion transport regulator; 2) is a major substrate for phosphorylation; and 3) very likely interacts with other proteins.

In rat hearts subjected to coronary ligation, application of cDNA microarrays (containing 86 known genes and 989 unknown cDNAs) to analyze transcript levels indicated that PLM was 1 of only 19 genes to increase after myocardial infarction (MI) (21). Specifically, when compared with sham-operated rat ventricles, PLM expression was increased twofold as early as 3 days after MI and remained elevated for at least 2 wk after MI. The effects of increased PLM on myocyte function are unknown. The present study was undertaken to evaluate whether PLM overexpression in normal adult rat cardiac myocytes alters contractile function and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) homeostasis in a manner similar to that observed in post-MI myocytes (3, 30, 31).

	METHODS

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PLM polyclonal antibody. Polyclonal antibody was raised against a 16-amino acid peptide fragment of the COOH terminus of PLM (NH₂-CGTFRSSIRRLSTRRR-COOH). To verify that the affinity-purified rabbit polyclonal PLM antibodies could indeed recognize PLM, we performed the following study. The coding sequence of canine heart PLM in pAlter-1 (a generous gift from Dr. J. R. Moorman, University of Virginia, Charlottesville, VA) was amplified by PCR with the following set of primers: 5'-GAA TTC CAT ATG GAA GCG CCA CAG GAA CAC-3' and 5'-AAG CTT CTC GAG CTA CTA CCG CCT GCG GGT-3'. The PCR product (246 bp) contained EcoRI and NdeI restriction sites at the 5' end and XhoI and HindIII restriction sites at the 3' end of the PLM sequence. After digestion with NdeI and XhoI, the PLM sequence was inserted into pET-19b, using the same NdeI and XhoI restriction sites on the cloning vector (Novagen, Madison, WI). The pET-19b vector contained an NH₂-terminal His tag sequence followed by an enterokinase site upstream of the NdeI and XhoI cloning sites. The cloned pET-19b-PLM plasmid was used to transform BL21(DE3)pLysS cells, and PLM expression was induced by isopropy1--D-thiogalactopyranoside. Bacterial lysate containing recombinant His-tagged PLM was mixed with Ni-NTA agarose slurry (Qiagen, Valencia, CA) and applied to a column, and His-tagged PLM was eluted and then dialyzed overnight against 2 M urea in phosphate-buffered saline. His-tagged PLM (1 µg/lane) in SDS sample buffer was subjected to 12% PAGE and transferred onto Immun-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). His-tagged PLM was detected with murine monoclonal anti-polyhistidine antibody (1:1,000 dilution, H1029; Sigma, St. Louis, MO), and sheep anti-mouse antibody (1:2,000, NA931; Amersham, Little Chalfont, UK) was used as the secondary antibody. Immunoreactive His-tagged PLM was detected with the enhanced chemiluminescence-Western blotting system (Amersham) and appeared as a single band of apparent molecular mass of ~22 kDa (Fig. 1, lane A). It is known that recombinant PLM exhibits anomalous migration on SDS polyacrylamide gels as an ~24-kDa protein (2). Overnight treatment of His-tagged PLM (1.8 µg) with recombinant enterokinase (1.6 U; Novagen) at room temperature to cleave off the His tag resulted in the expected loss of detection of His-tagged PLM by anti-polyhistidine antibody (Fig. 1, lane B). After the blot was stripped, the polyclonal antibody raised against the COOH terminus of PLM (1:5,000 dilution) was applied to the His-tagged PLM immunoblot and donkey anti-rabbit IgG (1:2,000, NA 934; Amersham) was used as the secondary antibody. Both the enterokinase-treated PLM (Fig. 1, lane C) and His-tagged PLM (Fig. 1, lane D) were recognized by our COOH terminus PLM antibody.

View larger version (4K): [in this window] [in a new window]   Fig. 1.   Polyclonal antibody to phospholamban (PLM). His-tagged PLM (1 µg/lane) from transformed bacterial lysates was eluted from Ni-NTA agarose column, dialyzed, subjected to 12% SDS-PAGE, and transferred onto Immun-Blot polyvinylidene difluoride (PVDF) membranes. A murine monoclonal anti-polyhistidine antibody (1:1,000) was used as the primary antibody to detect His-tagged PLM, which appeared as a single brand of apparent molecular mass of ~22 kDa on a Western blot (lane A). After cleavage of the His tag by enterokinase treatment, detection of His-tagged PLM by anti-polyhistidine antibody was lost (lane B). After the blot was stripped, application of the affinity-purified polyclonal antibody (1:5,000) against the COOH terminus of PLM to the blot resulted in detection of both enterokinase-treated PLM (lane C) and His-tagged PLM (lane D).

Myocyte isolation and culture. Cardiac myocytes were isolated from the septum and left ventricular free wall of male Sprague-Dawley rats (~280 g) by successive perfusion with collagenase and hyaluronidase (4). Portions of freshly isolated, Ca²⁺-tolerant myocytes were seeded on laminin-coated coverslips and used within 2 h of isolation for contractility measurements (5). For myocyte culture, myocytes were either seeded on laminin-coated coverslips, which were subsequently placed in four-well trays (Nuclone), or placed directly on laminin-coated four-well trays. Culture medium consisted of serum-free medium 199 (Earle's salts without L-glutamine and NaHCO₃) supplemented with (in mM) 25 NaHCO₃, 5 creatine, 5 taurine, 2 carnitine, and 0.1 ascorbic acid. Insulin (100 U/ml), 5'-bromo-2'-deoxyuridine (31 µg/ml), BSA (0.2%), penicillin (500 U/ml), and gentamicin (4 µg/ml) were also added to the culture medium. After 2 h, media were changed to remove nonadherent myocytes. The myocytes were incubated for an additional 3-4 h before initiation of electrical stimulation. Myocytes were electrically paced (1 Hz) in culture [extracellular Ca²⁺ concentration ([Ca²⁺]_o) = 1.8 mM] as previously described (32). Culture media were changed daily over the course of the experiments.

Construction of recombinant replication-deficient adenovirus. The basic protocol was described by He et al. (9). Briefly, the coding sequence of canine heart PLM (279 bp including 60 bp of signal sequence) together with 5'-untranslated (60 bp) and 3'-untranslated (200 bp) sequences were released from pAlter-1 by digestion with HindIII and EcoRI. The 571-bp fragment was then subcloned into pSP73 vector (Promega, Madison, WI) with the same HindIII and EcoRI restriction sites. The PLM sequence was released from pSP73 by digestion with HindIII and EcoRV and inserted into the shuttle vector pAdTrack-CMV, with the same HindIII and EcoRV restriction sites on the shuttle vector. The resulting shuttle vector plasmid was linearized with PmeI, mixed with supercoiled pAdEasy-1, and used to electroporate BJ5183 cells. The recombinants were identified by restriction endonuclease mapping (PacI). Once recombination was confirmed, supercoiled plasmid DNA was transformed into DH10B cells for large-scale amplification. The recombinant construct was linearized with PacI and used to transfect HEK-293 cells. Recombinant adenovirus expressing both green fluorescent protein (GFP) and PLM [each under a separate cytomegalovirus (CMV) promoter] (Adv-GFP-PLM) was harvested after 7 days, purified by CsCl gradient centrifugation, and stored at 20°C in 5 mM Tris (pH 8.0), 50 mM NaCl, 0.05% BSA, and 25% glycerol. The presence of the PLM coding sequence in recombinant adenovirus was confirmed by PCR with primers for the CMV promoter and the polyadenylation site of pAdTrack-CMV.

Adenoviral infection of cardiac myocytes. Two hours after isolation, myocytes seeded in four-well trays were infected with either Adv-GFP-PLM or adenovirus expressing GFP alone (Adv-GFP) at a multiplicity of infection of 2 for 3 h. Media were then changed, and myocytes were studied after 6, 24, 48, and 72 h in continued pacing culture. Over 95% of myocytes fluoresced green (excitation 478 nm, emission 535 nm) within 6 h, indicating successful adenoviral infection and GFP expression. For the sake of brevity, myocytes infected with Adv-GFP and Adv-GFP-PLM are referred to as GFP and PLM myocytes, respectively.

Myocyte shortening measurements. Myocytes adherent to the coverslips were bathed in 0.6 ml of air- and temperature (37°C)-equilibrated, HEPES-buffered (20 mM, pH 7.4) medium 199 containing 0.6, 1.8, or 5.0 mM [Ca²⁺]_o and were placed on a temperature-controlled stage (37°C) of a Ziess IM35 microscope. Measurements of myocyte contraction (1 Hz) were performed as previously described (28, 30, 32).

[Ca²+]_i transient measurements. Myocytes were exposed to 0.67 µM of fura 2-AM for 15 min at 37°C. Fura 2-loaded myocytes mounted in a Dvorak-Stotler chamber situated in a temperature-controlled stage (37°C) of a Zeiss IM35 inverted microscope were field stimulated to contract at 1 Hz between platinum wire electrodes (29, 31, 32). [Ca²⁺]_o was varied between 0.6 and 5.0 mM. [Ca²⁺]_i transient measurements, calibration of fura 2 fluorescence signals, and [Ca²⁺]_i transient analyses were performed as previously described (29, 31, 32).

PLM, Na+/Ca²⁺ exchanger, sarco(endo)plasmic reticulum Ca²⁺-ATPase, and calsequestrin immunoblotting. Cultured myocytes were harvested for immunoblotting on day 3. Cultured myocytes in a four-well tray were rinsed three times with ice-cold phosphate-buffered saline. They were then scraped into 1 ml of ice-cold lysis buffer containing (in mM) 50 Tris (pH 8.0), 150 NaCl, 1 Na⁺ orthovanadate, 1 phenylmethylsulfonyl fluoride, 100 NaF, 1 EDTA, and 1 EGTA, with 0.5% NP-40, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cell lysate was snap-frozen in dry ice-ethanol and stored at 80°C.

Statistics. All results are expressed as means ± SE. In experiments in which maximal contraction amplitudes were measured as a function of experimental group (Adv-GFP vs. +Adv-GFP), [Ca²⁺]_o, and days in culture, three-way ANOVA was performed to determine significance of difference. A linear model-fitted standard least squares (JMP version 4; SAS Institutes, Cary, NC) was used. For analysis of a parameter (e.g., systolic [Ca²⁺]_i, maximal contraction amplitude) as a function of group (GFP vs. PLM) and [Ca²⁺]_o, two-way ANOVA was used to determine statistical significance. Paired Student's t-test was used to compare protein abundance between GFP and PLM myocytes. In all analyses, P < 0.05 was taken to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of continual pacing and adenoviral infection on cultured myocyte contractility. We (27) and others (8) have demonstrated that adult rat cardiac myocytes cultured under quiescent conditions for as little as 6-18 h exhibited deterioration in cell shortening compared with freshly isolated myocytes. Maximal contraction amplitudes (1 Hz) of adult myocytes placed under continual pacing culture conditions were examined on days 0 (freshly isolated), 1, 2, and 3 and are shown in Fig. 2. Inspection of Fig. 2 suggests that for myocytes stimulated at 0.6 and 1.8 mM [Ca²⁺]_o, there were no differences in maximal contraction amplitudes on all 4 days. For myocytes stimulated at 5 mM [Ca²⁺]_o, maximal contraction amplitude declined modestly (from 17.70 ± 1.74 to 15.76 ± 3.51% resting cell length) after 3 days of culture. This conclusion is supported by two-way ANOVA ([Ca²⁺]_o, day in culture), which indicated significant [Ca²⁺]_o (P < 0.0001) and day in culture (P = 0.0174) effects. When data obtained at 5 mM [Ca²⁺]_o were removed from analysis, two-way ANOVA indicated significant [Ca²⁺]_o (P < 0.0001) effect, but time in culture had no effect on myocyte contraction amplitudes.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effects of continuous pacing culture and adenovirus infection on adult rat cardiac myocyte contractility. Isolated myocytes infected with recombinant adenovirus that expressed green fluorescent protein (GFP; filled symbols) were cultured at extracellular Ca2+ concentration ([Ca2+]o) of 1.8 mM for 72 h under continuous electrical stimulation (1 Hz) conditions (32). Myocytes not infected with adenovirus (open symbols) served as controls. For contraction studies, myocytes taken out of culture on different days were paced (1 Hz) to contract at 37°C and [Ca2+]o of 0.6 (circles), 1.8 (diamonds), and 5.0 (squares) mM. Each point represents the mean ± SE of 14-38 observations. Error bars are not shown if they fall within the boundaries of the symbol. Statistical analyses are given in RESULTS.

Effects of Adv-GFP-PLM infection on PLM abundance. Seventy-two hours after infection with either Adv-GFP or Adv-GFP-PLM, cardiac myocyte lysates were collected for analysis for PLM abundance by immunoblotting. Under reducing conditions, native cardiac PLM migrated on SDS-PAGE with an apparent molecular mass of 15-16 kDa (Fig. 3; Ref. 2). Compared with GFP myocytes, PLM myocytes had a significant 1.4-fold increase in PLM (Fig. 3; Table 1). By contrast, there were no significant differences in Na⁺/Ca²⁺ exchanger, SERCA2, or calsequestrin protein amounts between GFP and PLM myocytes (Fig. 3; Table 1).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Immunoblots of PLM, Na+/Ca2+ exchanger, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2), and calsequestrin. Proteins in myocyte homogenates (50 µg/lane) were separated by gel electrophoresis and transferred to Immun-Blot PVDF membranes, and PLM, Na+/Ca2+ exchanger, SERCA2, and calsequestrin were identified by immunoblotting as described in METHODS. Composite results are presented in Table 1. Numbers on left refer to apparent molecular mass.

Effects of PLM overexpression on contractile function. Preliminary studies indicated no significant differences in contraction amplitudes between GFP and PLM myocytes when they were examined 48 h after adenoviral infection (data not shown). Therefore, all contraction and [Ca²⁺]_i transient studies were performed after 72 h of adenoviral infection.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   PLM overexpression alters contractile function in adult rat myocytes. Isolated myocytes were infected with recombinant adenovirus expressing either GFP or both GFP and PLM and then cultured for 72 h under continuous electrical stimulation (1 Hz) conditions. For contraction studies, cultured myocytes were paced (1 Hz) to contract at 37°C and [Ca2+]o of 0.6, 1.8, or 5.0 mM. Shown are steady-state paced twitches from myocyte expressing either GFP (A, C, E) or PLM (B, D, F). Results are summarized in Table 2.

Effects of PLM overexpression on [Ca²+]_i transients. Resting [Ca²⁺]_i values in quiescent myocytes before stimulation were not different between GFP and PLM myocytes (group effect, P = 0.64) across the range of [Ca²⁺]_o examined (group × [Ca²⁺]_o effect, P = 0.57; Table 3). Similarly, end-diastolic [Ca²⁺]_i levels in myocytes paced at 1 Hz were not different (P = 0.80) between the two groups (Table 3). Varying [Ca²⁺]_o had no effect on either resting [Ca²⁺]_i ([Ca²⁺]_o effect, P = 0.85) or diastolic [Ca²⁺]_i ([Ca²⁺]_o effect, P = 0.58) in both GFP and PLM myocytes (Table 3).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   PLM overexpression alters cytosolic Ca2+ concentration ([Ca2+]i) transients in adult rat myocytes. Isolated myocytes were infected with recombinant adenovirus expressing either GFP (A, C, E) or both GFP and PLM (B, D, F) and then cultured for 72 h under continuous electrical stimulation (1 Hz) conditions. After being loaded with the Ca2+ indicator fura 2, myocytes were paced (1 Hz) to contract at 37°C and [Ca2+]o of 0.6, 1.8, and 5.0 mM. Results are summarized in Table 3.

	DISCUSSION

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Previous studies on PLM, a major substrate for protein kinases A and C in cardiac membranes (14, 18, 20), focused on its ion channel-like properties (16), channel regulatory roles (22), and cell volume regulatory function (17) in noncardiac cells. By contrast, there are few studies examining the potential function of PLM in the heart except for the interesting observation that PLM phosphorylation in response to adrenergic stimulation paralleled the positive inotropic effects (20). Recently, PLM mRNA was reported to increase twofold in postinfarction rat hearts (21). Because contractile function in postinfarction rat myocytes has been shown to be abnormal (3, 28, 30), we hypothesized that PLM might affect myocyte contractility by perturbing cardiac Ca²⁺ metabolism. To test this model, we have directed the overexpression of PLM in normal rat myocytes and measured its effects on contractility. We first established that under our continual pacing culture conditions, adult rat myocytes retained normal contractile function after 72 h of culture (Fig. 2), enough time for the exogenous PLM gene to be expressed and PLM protein to accumulate (Fig. 3). We also showed that the method of gene transfer by adenovirus infection had no effect on myocyte contractility (Fig. 2).

The first major finding of the present study is that PLM overexpression affected cardiac myocyte contractility (Fig. 4; Table 2). Specifically, under conditions that favored Ca²⁺ efflux (0.6 mM [Ca²⁺]_o), PLM myocytes contracted significantly more then GFP myocytes. Conversely, under conditions that favored Ca²⁺ influx (5.0 mM [Ca²⁺]_o), PLM myocytes shortened less than GFP myocytes. Assuming no changes in myofilament Ca²⁺ sensitivity with PLM overexpression, this complex pattern of contractile responses is consistent with the hypothesis that alterations in both Ca²⁺ influx and efflux pathways contribute to the different contractile behavior in PLM myocytes. Interestingly, the pattern of contractile abnormalities in PLM myocytes resembles that observed in postinfarction rat myocytes in that compared with sham-operated controls, contraction amplitudes in MI myocytes were higher at 0.6 mM but lower at 5.0 mM [Ca²⁺]_o (28, 30). Thus our observations are consistent with the hypothesis that overexpression of PLM after infarction (21) may be causally related to development of contractile dysfunction.

If the differences in contractile behavior between GFP and PLM myocytes were in fact due to altered Ca²⁺ fluxes with PLM overexpression, then changes in [Ca²⁺]_i transients during a stimulated twitch should be observed. This is indeed the case (Fig. 5; Table 3), and it constitutes the second major finding of the study. Similar to twitch amplitudes, compared with GFP myocytes, [Ca²⁺]_i transient amplitudes were higher at 0.6 mM [Ca²⁺]_o but lower at 5.0 mM [Ca²⁺]_o. The observation that [Ca²⁺]_i transient amplitude differences between GFP and PLM myocytes mirror the pattern of contractile behavior differences supports our hypothesis that the abnormal contraction was caused by perturbed Ca²⁺ fluxes associated with PLM overexpression.

Overexpression of PLM was not associated with changes in protein levels of major cardiac Ca²⁺ transporters. Specifically, the levels of Na⁺/Ca²⁺ exchanger, SERCA2, and calsequestrin were similar in GFP and PLM myocytes. In addition, SR Ca²⁺ uptake activity, as estimated by t_1/2 of [Ca²⁺]_i decline (26, 31), was not affected by PLM overexpression.

The mechanisms by which PLM perturbed Ca²⁺ fluxes and contractile function have not been addressed in the present exploratory study. However, the known properties of PLM measured in noncardiac cells provide a reasonable basis for speculation. PLM forms channels that are permeable to the zwitterion taurine (16, 17). Thus overexpression of PLM may lead to loss of taurine from cardiac myocytes. Taurine depletion is associated with myocardial contractile dysfunction (19), probably due to disordered contractile filaments and loss of myofibrillar bundles (13). However, the deleterious effects of taurine depletion on cardiac contractile elements should lead to reduction in contraction amplitudes at all [Ca²⁺]_o levels (7). This is clearly not the case, because in PLM myocytes contraction amplitudes measured at 0.6 mM [Ca²⁺]_o were higher than in GFP myocytes (Fig. 4; Table 3). An alternative mechanism by which taurine may affect myocyte contractility is perturbation of [Ca²⁺]_i homeostasis by changing Ca²⁺ fluxes through the Na⁺/Ca²⁺ exchanger (1). However, the effects of taurine on Na⁺/Ca²⁺ exchanger function remain controversial (1, 7, 11).

Another potential mechanism by which PLM can affect contractility is by inhibiting Na⁺/Ca²⁺ exchange activity. The pattern of contractile and [Ca²⁺]_i transient abnormalities observed in PLM myocytes (amplitudes higher at 0.6 mM [Ca²⁺]_o but lower at 5.0 mM [Ca²⁺]_o) mimics that observed in postinfarction rat myocytes (3, 28-31), in which Na⁺/Ca²⁺ exchange activity has been shown to be depressed (6, 33). In addition, the pattern of contraction and [Ca²⁺]_i transient alterations in PLM myocytes is opposite to that observed in myocytes in which Na⁺/Ca²⁺ exchange activity was enhanced by overexpression (32). Specifically, in adult rat myocytes overexpressing the Na⁺/Ca²⁺ exchanger, both contraction and [Ca²⁺]_i transient amplitudes were lower at 0.6 mM [Ca²⁺]_o but higher at 5.0 mM [Ca²⁺]_o compared with their controls (32). These two observations suggest that PLM probably regulates Na⁺/Ca²⁺ exchange activity, either directly or indirectly.

PLM belongs to the FXYD gene family of small ion transport regulators (23). This family includes the -subunit of Na⁺-K⁺-ATPase. The -subunit of Na⁺-K⁺-ATPase has been shown to bind to and modulate Na⁺-K⁺-ATPase activity (24). A potential mechanism by which PLM alters myocyte contraction is inhibition of Na⁺-K⁺-ATPase activity, thereby indirectly modulating Na⁺/Ca²⁺ exchange activity. Further support for this hypothesis is the observation that shark rectal glands contained a PLM-like protein (PLMS) that associated with the -subunit of Na⁺-K⁺-ATPase (15). PLMS has homology to PLM and similar mobility on SDS-PAGE of 15 kDa (15). Unlike the -subunit of Na⁺-K⁺-ATPase, which lacked PLM's basic COOH-terminal sequence with phosphorylation motifs (18), PLMS could be phosphorylated by both protein kinase A and protein kinase C (15). Phosphorylated PLMS dissociated from the -subunit of Na⁺-K⁺-ATPase, with resultant activation of the enzyme (15). The close homology between PLM and PLMS suggests that PLM may modulate Na⁺-K⁺-ATPase activity by association with the enzyme in cardiac myocytes, similar to the PLMS association with shark Na⁺-K⁺-ATPase. Thus it is possible that PLM could modulate Na⁺-K⁺-ATPase activity and thus indirectly affect Na⁺/Ca²⁺ exchange. Inhibition of Na⁺-K⁺-ATPase by PLM would be expected to increase intracellular Na⁺ and, based on thermodynamics considerations alone, should diminish forward Na⁺/Ca²⁺ exchange (Ca²⁺ efflux) but enhance reverse Na⁺/Ca²⁺ exchange (Ca²⁺ influx). This would result in increased contraction amplitudes in PLM myocytes studied under both low (Ca²⁺ efflux-promoting)- and high (Ca²⁺ influx promoting)-[Ca²⁺]_o conditions, a prediction not consistent with our observations on myocytes overexpressing PLM. Therefore, mechanisms other than or in addition to sole inhibition of Na⁺-K⁺-ATPase by PLM needed be invoked to fully explain our experimental observations. In this light, we should not overlook the possibility that PLM directly interacts with Na⁺/Ca²⁺ exchanger and modulates its activity. Much further work is required, however, to elucidate the mechanisms by which PLM affects cardiac contractility.

In summary, we have established an in vitro myocyte culture model system in which normal contractile function was preserved for at least 72 h. Infection with adenovirus had no effect on myocyte contractility. Overexpression of PLM by adenovirus-mediated gene delivery resulted in altered contraction and [Ca²⁺]_i transients. Specifically, PLM myocytes contracted more than GFP myocytes at 0.6 mM [Ca²⁺]_o but shortened less than GFP myocytes at 5.0 mM [Ca²⁺]_o. This pattern of contractile abnormality is similar to that observed in postinfarction myocytes, in which contraction amplitudes were higher at 0.6 mM [Ca²⁺]_o but lower at 5.0 mM [Ca²⁺]_o compared with sham-operated myocytes (30). Na⁺/Ca²⁺ exchanger, calsequestrin, and SERCA2 expression and SR Ca²⁺ uptake activity were not affected by PLM overexpression. We conclude that overexpression of PLM resulted in perturbation of Ca²⁺ fluxes, with resultant contractile abnormalities in adult rat myocytes. We speculate that PLM overexpression may partly account for the contractile dysfunction in postinfarction myocytes.