INTRODUCTION

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PHOSPHOLEMMAN (PLM) is a 72-amino acid membrane phosphoprotein with a single transmembrane domain. In heart and skeletal muscle, PLM is phosphorylated in response to adrenergic (16, 22) and insulin (30) stimulation. PLM is also a substrate for myotonic dystrophy protein kinase (19). The physiological function of PLM in the heart is largely unknown except that PLM phosphorylation in response to adrenergic stimulation paralleled the positive inotropic effects (22) and that expression of PLM increased twofold in postinfarction rat hearts (25). In a previous study, we (27) have demonstrated that PLM overexpression in adult rat myocytes resulted in altered contractility and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients. Specifically, at low extracellular [Ca²⁺] ([Ca²⁺]_o; 0.6 mM), both contraction and [Ca²⁺]_i transient amplitudes were larger in myocytes overexpressing PLM. At high [Ca²⁺]_o (5 mM), cell shortening and [Ca²⁺]_i transient amplitudes were reduced in myocytes overexpressing PLM. This pattern of contractile and [Ca²⁺]_i transient abnormalities observed in myocytes overexpressing PLM mimics that observed in postinfarction rat myocytes (3, 31), in which Na⁺/Ca²⁺ exchanger (NCX1) activity has been shown to be depressed (6, 33). In addition, downregulation of NCX1 in rat myocytes by adenovirus (Adv)-mediated antisense transfer resulted in a pattern of contraction and [Ca²⁺]_i transient abnormalities (29) similar to that observed in myocytes overexpressing PLM (27). Finally, the pattern of [Ca²⁺]_i transient and contraction abnormalities in myocytes overexpressing PLM (27) was opposite to that observed in myocytes in which NCX1 activity was enhanced by overexpression (32). These observations strongly suggest, but do not prove, that PLM affects cardiac myocyte contractility by modulating Na⁺/Ca²⁺ exchange activity. The present study was undertaken to test the hypothesis that PLM directly affects Na⁺/Ca²⁺ exchange activity in rat cardiac myocytes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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). Isolated 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 media 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), bovine serum albumin (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 in culture (1 Hz, [Ca²⁺]_o = 1.8 mM) as previously described (32). Culture media were changed daily over the course of the experiments. Under continuous pacing culture conditions, we have (27) previously demonstrated that myocyte contractility did not decline for at least 72 h.

Adenoviral infection of cardiac myocytes. Recombinant, replication-deficient Adv expressing either green fluorescent protein (GFP) alone, GFP and PLM (27), or GFP and NCX1 (32) were constructed as described previously. Two hours after isolation, myocytes seeded in four-well trays were infected with Adv-GFP, Adv-GFP-PLM, or Adv-GFP-NCX1 plus Adv-GFP-PLM at a multiplicity of infection of 2 for 3 h. Media were then changed, and myocytes were studied after 72 h in continued pacing culture. We have previously demonstrated that over 95% of myocytes were successfully infected (32) and that adenoviral infection of myocytes had no effects on myocyte contractility when examined after 72 h of continuous pacing culture (27). We have also previously shown that the effects of PLM overexpression on contractility and [Ca²⁺]_i transients were manifest 72 h after Adv-PLM infection (27). For the sake of brevity, myocytes infected with Adv-GFP, Adv-GFP-PLM, and Adv-GFP-NCX1 plus Adv-GFP-PLM are referred to as GFP, PLM, and PLM-NCX1 myocytes, respectively.

Myocyte shortening measurements. Myocyte adherent to the coverslips were bathed in 0.6 ml of air- and temperature (37°C)-equilibrated, HEPES-buffered (20 mM, pH 7.4) media 199 containing either 0.6 or 5.0 mM [Ca²⁺]_o and were placed on a temperature-controlled stage (37°C) of a Zeiss IM35 microscope. Measurements of myocyte contraction (1 Hz) were performed as previously described (27, 29, 31, 32).

Caffeine-induced contractures. Myocytes bathed in 5.0 mM [Ca²⁺]_o were paced at 1 Hz. At 200 ms after the 21st beat, a trigger TTL pulse of 2.4-s duration was generated by a programmable multichannel stimulator (STIM-6, Ionoptix; Milton, MA) to initiate caffeine (5 mM) application by puffer (Ionoptix) superfusion, which allowed rapid solution changes around a single cell (29, 31-33). The caffeine-induced contracture and subsequent relaxation were captured by a charge-coupled device videocamera (Ionoptix) and analyzed (29, 31).

Na+/Ca²⁺ exchange current measurements. Whole cell patch-clamp recordings were performed at 30°C as described previously (29, 33, 34). Briefly, fire-polished pipettes (tip diameter 4-6 µm) with resistances of 0.8-1.4 M when filled with standard internal solution were used. To facilitate reverse-mode operation of NCX1, pipettes were filled with a nominally Ca²⁺-free solution containing (in mM) 100 Cs⁺ glutamate, 7.25 NaCl, 1 MgCl₂, 20 HEPES, and 2.5 Na₂ ATP; pH 7.2. EGTA was normally not included in the pipette filling solution. Free Ca²⁺ in the EGTA-free pipette solution was 187 nM, measured fluorimetrically with fura 2. In another series of experiments, EGTA (10 mM) and CaCl₂ (6 mM) were added to the pipette filling solution such that free Ca²⁺ was "clamped" at 205 nM (verified fluorimetrically). To maintain similar osmolarities between EGTA-containing and EGTA-free pipette solutions, Cs⁺ glutamate was reduced from 100 to 80 mM in EGTA-containing solutions.

Action potential measurements. Action potentials (APs) from GFP and PLM myocytes were recorded after 72 h of continual pacing culture using the current-clamp configuration at 1.5 times the threshold stimulus and 4-ms duration (29, 34). Pipette solution consisted of (in mM) 125 KCl, 4 MgCl₂, 0.06 CaCl₂, 10 HEPES, 5 potassium EGTA, 3.1 Na₂ ATP, and 5 Na₂-creatine phosphate; pH 7.2. External solution consisted of (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 0.6 NaH₂PO₄, 15 HEPES, and 5 glucose; pH 7.4.

NCX1, PLM, 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 PBS. 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 PMSF, 100 NaF, 1 EDTA, and 1 EGTA, and 0.5% NP-40, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cell lysate was snap frozen with dry ice-ethanol and stored at 80°C.

Immunolocalization of PLM and NCX1 in adult rat cardiac myocytes. Freshly isolated rat cardiac myocytes were plated on laminin-coated glass slide chambers (Nunc, Lab-Tek Division; Naperville, IL) and allowed to adhere for 2 h. Adherent myocytes were chilled on ice and rinsed three times with ice-cold PBS containing 2 mM EGTA. Myocytes were fixed for 30 min on ice in cold 3% paraformaldehyde in PBS with 2 mM EGTA. After two rinses with ice-cold PBS, cells were permeabilized for 2 min in cold 0.05% Triton X-100. Permeabilized myocytes were rinsed two times with PBS and one time with BLOTTO (5% nonfat dry milk, 0.1 M NaCl, and 50 mM Tris · HCl; pH 7.4). Primary antibodies against NCX1 (1:100, R3F1, mouse monoclonal) and PLM (1:100, rabbit polyclonal) diluted in BLOTTO were added to the cells, incubated at room temperature in the dark for 60 min, and rinsed three times with BLOTTO. Secondary antibodies (1:200, see below) diluted in BLOTTO were added to cells that were incubated in the dark for 30 min, followed by three PBS rinses. The slide was then removed from the chamber, and a coverslip containing mounting solution (90% glycerol in PBS + p-phenylaminediamine) was applied. Myocytes were viewed with a Nikon Optiphot-2 microscope equipped for epifluorescence. Images were acquired with an air-cooled charge-coupled device SenSys digital camera (Photometrics; Tucson, AZ) and processed using IPLab and Enhanced Photon Reassignment software (Scanalytics; Fairfax, VA). Secondary antibodies used were as follows: goat anti-rabbit IgG conjugated with Texas red-X (Molecular Probes; Eugene, OR), goat anti-mouse IgG coupled to FITC (Sigma; St. Louis, MO), donkey anti-mouse IgG conjugated with Texas red, and donkey anti-rabbit IgG coupled to FITC (Jackson ImmunoResearch; West Grove, PA).

Statistics. All results are expressed as means ± SE. For analysis of I_NaCa as a function of group (GFP vs. PLM) and test voltage, two-way ANOVA was used to determine statistical significance. For analysis of contraction amplitudes as a function of group (GFP vs. PLM vs. PLM-NCX1) and [Ca²⁺]_o, two-way ANOVA was used. For analysis of AP parameters, caffeine contracture relaxation, and protein abundance, Student's t-test was used. A commercial software package (JMP version 4, SAS Institutes; Cary, NC) was used in all statistical analyses. In all analyses, P < 0.05 was taken to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of PLM overexpression on relaxation from caffeine-induced contractures. After steady-state twitch amplitude was achieved, application of 5 mM caffeine to a myocyte at end diastole caused a large contracture due to sarcoplasmic reticulum (SR) Ca²⁺ release and then relaxation to a shorter resting cell length in the continued presence of caffeine (Fig. 1). The incomplete relaxation is thought to be due to increased myofilament sensitivity to Ca²⁺ by caffeine. Relaxation in the continued presence of caffeine was mediated primarily by forward Na⁺/Ca²⁺ exchange, because SR Ca²⁺ accumulation was inhibited (33). The half-time of relaxation from caffeine-induced contracture was significantly (P < 0.003) slower in PLM (2.90 ± 0.29s, n = 11) than GFP (1.68 ± 0.23s, n = 14) myocytes (Fig. 1), suggesting reduced forward Na⁺/Ca²⁺ exchange activity in PLM myocytes.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Overexpression of phospholemman (PLM) slows relaxation from caffeine-induced contractures in adult rat myocytes. After 21 twitches [1 Hz, 37°C, 5 mM extracellular [Ca2+] ([Ca2+]o)] to ensure steady-state sarcoplasmic reticulum (SR) Ca2+ load, caffeine (5 mM) was "puffed" onto a myocyte for 2.4 s to induce contracture, and myocyte relaxation was followed until complete. We (33) have previously shown that 2.4 s of caffeine exposure was sufficient to release all SR Ca2+ contents in rat myocytes. At low (5 mM) caffeine concentrations, rapid transient phase of contraction largely reflects changes in intracellular [Ca2+] ([Ca2+]i). Relaxation in continued presence of caffeine was primarily due to forward Na+/Ca2+ exchange because SR Ca2+ accumulation was inhibited. A: myocytes infected with adenovirus expressing green fluorescent protein (GFP); B: myocytes infected with adenovirus expressing both GFP and dog heart PLM. Myocytes were studied 72 h after infection.

Effects of PLM overexpression on reverse I_NaCa and cell size in adult rat myocytes. Figure 2 shows the steady-state reverse I_NaCa measured at various membrane potentials, at 30°C and 5.0 mM [Ca²⁺]_o, in both GFP (open symbols; n = 17) and PLM (filled symbols; n = 16) myocytes in which [Ca²⁺]_i (187 nM) was not buffered. It can be appreciated that the absolute magnitudes of I_NaCa were in general lower in PLM than GFP myocytes. Two-way ANOVA confirmed a significant group (PLM vs. GFP) effect (P < 0.0003). In both GFP and PLM myocytes, depolarization to more positive membrane potentials increased the absolute magnitude of I_NaCa (voltage effect, P < 0.0001). In addition, the differences in I_NaCa between GFP and PLM myocytes were amplified at more positive membrane voltages (group × voltage interaction effect, P < 0.0001).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Current-voltage relationships of reverse Na+/Ca2+ exchange current (INaCa) in GFP and PLM myocytes. A: 72 h after adenovirus infection, leak-subtracted steady-state INaCa at each test potential was measured as described in METHODS. B: means ± SE for 17 GFP () and 16 PLM () myocytes incubated at 5 mM [Ca2+]o and 30°C are shown. Free [Ca2+] in the pipette solution was 187 nM. Error bars are not shown if they fall within boundaries of symbol.

Effects of PLM overexpression on AP. Changes in I_NaCa in PLM myocytes may potentially affect AP amplitude and duration. Therefore, we measured AP in GFP and PLM myocytes (Fig. 3). Resting membrane potential (RMP), AP amplitude, and AP duration at 50% repolarization measured in GFP myocytes (Fig. 3 and Table 1) were similar to those reported for adult rat myocytes cultured in serum-free media for 72 h (7). There were no significant differences in RMP, AP amplitude, and AP duration at 50% and 90% repolarization between GFP and PLM myocytes (Fig. 3 and Table 1), indicating that PLM overexpression had no effect on myocyte RMP and AP morphology.

View larger version (6K): [in this window] [in a new window]   Fig. 3.   Overexpression of PLM does not affect action potential amplitude and duration in adult rat myocytes. Seventy-two hours after adenovirus exposure, action potentials in GFP (A) and PLM (B) myocytes were measured at 30°C, as described in METHODS. Composite data are summarized in Table 1.

Cooverexpression of NCX1 corrects contractile abnormalities in PLM myocytes. We have previously shown (27) that compared with GFP myocytes, maximal cell shortening amplitudes measured at 0.6 mM [Ca²⁺]_o were higher but those measured at 5 mM [Ca²⁺]_o were lower in PLM myocytes. We have confirmed this finding in the present study (Fig. 4, A-D, and Table 2). More importantly, in myocytes in which both PLM and NCX1 were overexpressed (Fig. 5), maximal shortening amplitudes at 5 mM [Ca²⁺]_o were increased toward control GFP myocyte levels (Fig. 4, B, D, and F, and Table 2). At 0.6 mM [Ca²⁺]_o, the supranormal contraction amplitudes of PLM myocytes (Fig. 4C) were reduced toward control GFP levels (Fig. 4A) by cooverexpression with NCX1 (Fig. 4E) (Table 2). In all three groups, increasing [Ca²⁺]_o increased maximal contraction amplitudes (Table 2). These conclusions are supported by highly significant (P < 0.0001) [Ca²⁺]_o and group × [Ca²⁺]_o interaction effects in maximal contraction amplitudes between GFP and PLM myocytes, indicating the magnitude and/or direction of the effects of [Ca²⁺]_o on cell shortening was different between GFP and PLM myocytes. Likewise, comparison between PLM and PLM-NCX1 myocytes demonstrated significant (P  0.0001) [Ca²⁺]_o and group × [Ca²⁺]_o interaction effects. By contrast, there were no significant (P > 0.12) group × [Ca²⁺]_o interaction effects between GFP and PLM-NCX1 myocytes, although the [Ca²⁺]_o effect was still significant (P < 0.0001).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Cooverexpression of Na+/Ca2+ exchanger (NCX1) ameliorates contractile abnormalities in PLM myocytes. Isolated myocytes were infected with recombinant adenovirus expressing either GFP alone, or GFP and PLM, or GFP and NCX1 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 (A, C, and E) or 5 mM (B, D, and F). Shown are steady-state paced twitches from myocytes expressing either GFP (A and B), PLM (C and D), or PLM and NCX1 (E and F). Results are summarized in Table 2.

View larger version (5K): [in this window] [in a new window]   Fig. 5.   Immunoblots of NCX1, PLM, and calsequestrin. Proteins in myocyte homogenates (5 µg/lane) were separated by gel electrophoresis and transferred to ImmunoBlot polyvinylidene difluoride membranes, and NCX1, PLM, and calsequestrin were identified by immunoblotting, as described in METHODS. Composite results are presented in Table 3. Numbers on the left refer to apparent molecular mass.

Immunofluorescence localization of PLM and NCX1 in adult rat myocytes. The specificities of monoclonal anti-NCX1 antibody R3F1 (21) and polyclonal anti-PLM antibody (27) have been detailed elsewhere. Under nonreducing conditions, R3F1 recognized a single band with an apparent molecular mass of 160 kDa in rat myocytes homogenates (Fig. 5), which corresponded to NCX1 (20). The polyclonal antibodies raised against the COOH-terminus of PLM recognized a single band with apparent molecular mass of 15-16 kDa (Fig. 5), where native cardiac PLM was expected to migrate on SDS-PAGE (2). Both PLM (Fig. 6A) and NCX1 (Fig. 6B) antibodies labeled adult rat ventricular myocytes extensively in all regions exposed to the extracellular space: at the sarcolemma, at intercalated disks, and in the transverse tubules (t tubules). Images at different planes through the myocyte (0.25-µm sections, ~85 sections/myocyte) were then deconvolved to remove out-of-focus contaminating light to generate high-resolution images (Fig. 6, D, E, G, and H). These images from the centers of myocytes revealed similar features that were identified in the wide-field views (Fig. 6, A and B). In particular, no specific label was detected in intracellular structures or organelles. In addition, when the stacks of PLM and NCX1 images were processed and merged (Fig. 6, F and I), it is apparent that PLM and NCX1 colocalized to the same areas of the myocyte. This is true whether PLM was detected with FITC-labeled secondary antibody and NCX1 with Texas red-labeled secondary antibody (Fig. 6, D-F) or whether PLM was detected with Texas red-labeled secondary antibody and NCX1 with FITC-labeled secondary antibody (Fig. 6, G-I).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Immunofluorescence localization of PLM and NCX1. Indirect immunofluorescence of adult rat ventricular myocytes double labeled with mouse monoclonal anti-NCX1 antibody (R3F1) (B, E, and H) and rabbit polyclonal anti-PLM antibody (27) (A, D, and G) are shown. Primary antibodies were visualized with FITC-labeled donkey anti-rabbit IgG (D), Texas red-conjugated donkey anti-mouse IgG (E), Texas red-labeled goat anti-rabbit IgG (G), and FITC-labeled goat anti-mouse IgG (H). A and B: wide-field immunofluorescence images. D, E, G, and H: deconvolved images taken at the center of the myocytes. C, F, and I: merged images of A and B, D and E, and G and H, respectively. Note the orange color in the merged images, suggesting colocalization of NCX1 and PLM. Bar = 5 µm.

	DISCUSSION

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PLM belongs to the FXYD gene family of small ion transport regulators (28). In noncardiac tissues, PLM has been shown to exhibit ion channel-like behavior (17), channel regulatory roles (26), and osmotic regulatory function (18). In adult rat cardiac myocytes, we have recently demonstrated that PLM overexpression resulted in altered contraction and [Ca²⁺]_i transients, without affecting NCX1 and SR Ca²⁺-ATPase protein levels and SR Ca²⁺ uptake (27). The mechanisms by which PLM perturbed Ca²⁺ fluxes and contractile function, however, are not known. Because 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 mM [Ca²⁺]_o) mimicked that observed in myocytes in which Na⁺/Ca²⁺ exchange activity was depressed (29, 31, 33) and was opposite to that observed in NCX1-overexpressed myocytes (32), we speculated that PLM might directly regulate Na⁺/Ca²⁺ exchange activity. Thus the first major finding of the present study is that PLM overexpression directly inhibited Na⁺/Ca²⁺ exchange activity, both in the forward mode (3 Na⁺ in:1 Ca²⁺ out), as indicated by prolonged relaxation from caffeine-induced contractures (Fig. 1), and in the reverse mode (3 Na⁺ out:1 Ca²⁺ in), as evidenced by reduced reverse I_NaCa (Fig. 2).

Our present I_NaCa values (measured at + 70 mV) in 3-day cultured myocytes were 59% lower when compared with those previously measured in freshly isolated rat cardiac myocytes (33). The differences may be due to the effects of short-term culture, but more likely relate to the fact that we used a more physiological but lower pipette [Na⁺] (12.5 vs. 25 mM) in the present experiments. The 28-33% difference in I_NaCa between control GFP and PLM myocytes, although modest, is likely to be physiologically relevant because relaxation from caffeine-induced contracture was slower (Fig. 1) and myocyte contraction was affected in PLM myocytes (Fig. 4 and Table 2). In this context, the difference in I_NaCa between GFP and PLM myocytes is in the same order of magnitude as that between sham and postinfarction rat myocytes (46%; Ref. 33); the latter has been demonstrated to suffer from contraction abnormalities and slowed relaxation from caffeine-induced contractures (31).

In the present study, we confirmed our previous observation (27) that PLM overexpression resulted in increased contraction amplitude at 0.6 mM [Ca²⁺]_o but decreased cell shortening at 5 mM [Ca²⁺]_o (Fig. 4, A-D, and Table 2). Because depressed I_NaCa in PLM-overexpressed myocytes (Fig. 2) may alter AP morphology, the abnormal pattern of contraction in PLM myocytes could be due to AP changes rather than directly resulting from reduced NCX1 function. Thus a second major finding is that PLM overexpression did not affect RMP and AP morphology in adult rat myocytes (Fig. 3 and Table 1). In addition, the pattern of contractile abnormalities observed in PLM myocytes could be ameliorated by cooverexpression of NCX1 (Fig. 4 and Table 2), thereby providing additional support, albeit indirect, that PLM affected myocyte contractile function by modulating Na⁺/Ca²⁺ exchange activity.

In addition to thermodynamic parameters [intracellular ([Na⁺]_i) and extracellular [Na⁺] ([Na⁺]_o), [Ca²⁺]_i, [Ca²⁺]_o, and membrane potential) that regulate Na⁺/Ca²⁺ exchange activity, other known factors that enhance NCX1 activity include ATP/phosphatidyl inositol 4,5-bisphosphate, acidic phospholipids, phosphoarginine, and protein kinases A and C (1, 8, 10, 13). Inhibitory factors of NCX1 activity include extracellular [Mg²⁺], acidic pH, XIP peptide, sulfhydryl groups, and an unknown endogenous inhibitor of 350-550 kDa (1, 11, 15, 23). Our present results (Figs. 1, 2, and 4) indicate that one of the physiological functions of PLM in cardiac tissues is to modulate NCX1 activity. Our results, however, did not differentiate whether phosphorylated or dephosphorylated PLM is required for its inhibitory effects on NCX1 activity. On the basis of the observation that PLM phosphorylation in response to adrenergic stimulation paralleled the inotropic response (22), one may speculate that NCX1 activity is inhibited by PLM, with which, on phosphorylation of PLM by protein kinases A (16, 22) or C (30), the inhibitory effect is removed. In this light, it is interesting to note that enhanced NCX1 activity by protein kinase C (13) did not require direct phosphorylation of the NCX1 (12).

The mechanisms by which PLM inhibits NCX1 activity have not been addressed in this study. Recent coimmunoprecipitation studies indicate that PLM associates with -isozymes of Na⁺-K⁺-ATPase in bovine cardiac sarcolemma (5). In addition, when coexpressed with rat Na⁺-K⁺-ATPase (either ₁-₁ or ₂-₁ isozymes) in Xenopus oocytes, PLM effected a 1.5- to 2-fold decrease in the apparent affinity of Na⁺-K⁺-ATPase for internal Na⁺ (5). Inhibition of Na⁺-K⁺-ATPase by PLM would result in elevation in [Na⁺]_i and theoretically could modulate NCX1 activity indirectly. This mechanism is unlikely to account for our experimental observations on PLM myocytes for the following reasons. On the basis of thermodynamics considerations alone, increased [Na⁺]_i 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 in PLM myocytes (Fig. 4 and Table 2). In addition, under our whole cell patch-clamp conditions, in which [Na⁺]_i in the well-dialyzed myocyte was likely to be "clamped" at pipette [Na⁺] and Na⁺-K⁺-ATPase activity would be minimal in the absence of K⁺ in both external and pipette filling solutions, reverse I_NaCa was still significantly lower in PLM than GFP myocytes (Fig. 2).

Another major finding of the present study is that PLM was detected only in the sarcolemma, intercalated disks, and t tubules (Fig. 6, A, D, and G). This, we believe, is the first report of PLM localization in cardiac tissues. We also confirmed previous reports (9, 14, 24) that NCX1 was localized to the t tubules and sarcolemma (Fig. 6, B, E, and H), although there is some controversy as to the relative distribution of NCX1 in adult myocytes (9, 14). More interestingly, the merged images (Fig. 6, C, F, and I) suggest colocalization of PLM and NCX1 to the same membrane regions of the myocyte, thus providing anatomic support for interactions between PLM and NCX1. We wanted to emphasize that immunofluorescence colocalization of two molecules does not prove direct interaction between the two molecules. Higher resolution techniques such as fluorescence resonance energy transfer and yeast two-hybrid systems are better suited to detect molecular interactions.

In myocytes isolated from rat hearts 3-8 wk after myocardial infarction (MI), both reverse I_NaCa (33) and Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles (6) were depressed, and relaxation from caffeine-induced contracture was prolonged (31). In addition, when compared with myocytes isolated from sham-operated hearts, twitch amplitudes were higher at 0.6 mM [Ca²⁺]_o but lower at 5 mM [Ca²⁺]_o in post-MI myocytes (31). It has been shown that PLM expression was increased twofold post-MI (25). An interesting speculation is that at least some of the contractile abnormalities observed in post-MI myocytes is due to enhanced PLM expression, leading to increased inhibition of Na⁺/Ca²⁺ exchange activity, with resultant alteration in contractile function.

In summary, we have demonstrated direct inhibition of both forward and reverse Na⁺/Ca²⁺ exchange activity by PLM overexpression in adult rat myocytes. Contractile abnormalities in PLM myocytes were ameliorated by cooverexpression of NCX1. Immunofluorescence studies suggest colocalization of PLM and NCX1 to the sarcolemma, intercalated disks, and t-tubules. We conclude that PLM affects myocyte contractility by modulating Na⁺/Ca²⁺ exchanger function. We speculate that overexpression of PLM may partly account for contractile abnormalities in postinfarction myocytes.