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1Weis Center for Research and 2Department of Medicine, Geisinger Medical Center, Danville, Pennsylvania 17822; and 3Division of Cardiology, Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville, Virginia 22908
Submitted 23 October 2003 ; accepted in final form 12 December 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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adult rat myocyte culture; patch clamp; fura-2; edge detection; excitation-contraction coupling
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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280 g) as previously described (3). Isolated myocytes were seeded on laminin-coated coverslips and cultured with serum-free medum 199 (Earle's salts without L-glutamine and NaHCO3) supplemented with creatine, carnitine, taurine, and NaHCO3 (17, 26). After 2 h, media were changed to remove nonadherent myocytes. Six hours after isolation, cultured myocytes were electrically paced {1 Hz, extracellular Ca2+ concentration ([Ca2+]o) = 1.8 mM} (17, 26). Culture media were changed daily over the course of experiments. Under continuous pacing culture conditions, we have previously demonstrated that myocyte contractility did not decline for at least 72 h (17). Construction of recombinant replication-deficient adenovirus expressing antisense PLM. The basic protocol has been described by He et al. (7). Initially, the coding sequence of dog heart PLM together with 5'-untranslated and 3'-untranslated sequences (13, 17) was released from pAlter-1 (Promega; Madison, WI) by digestion with HindIII and SalI. The dog PLM fragment was inserted in the antisense (AS) direction into the shuttle vector pAdTrack-CMV (this vector, plus BJ5183 and DH10B cells, were generous gifts from Dr. B. Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD) with SalI and HindIII restriction sites. The resulting shuttle vector plasmid was linearized with PmeI, mixed with supercoiled pAdEasy-1, and used to electroporate BJ5183 cells. The recombinant plasmids were selected and screened by restriction endonuclease mapping (PacI). Once recombination was confirmed, supercoiled plasmid DNA (isolated from minipreps) was transformed into DH10B cells for large-scale amplification. The recombinant construct was linearized with PacI and used to transfect HEK-293 cells (Clontech; Palo Alto, CA). Recombinant Adv expressing both green fluorescent protein (GFP) and dog AS PLM [dASPLM; each under a separate cytomegalovirus (CMV) promoter] (Adv-GFP-dASPLM) were harvested at 7 days, purified on a CsCl gradient, and stored at –20°C in 5 mM Tris (pH 8.0), 50 mM NaCl, 0.05% bovine serum albumin, and 25% glycerol. The presence of dASPLM in recombinant Adv was confirmed by PCR, using primers for the CMV promoter and the polyadenylation site of pAdTrack-CMV.
In the majority of experiments, a recombinant Adv expressing GFP and rat AS PLM (rASPLM) was constructed and used. Briefly, 100 mg of rat LV tissue were homogenized in 1 ml TRIzol reagent (GIBCO-BRL; Grand Island, NY). Chloroform (0.2 ml) was added to the homogenate, and, after centrifugation (2,600 g, 30 min), RNA was ethanol precipitated from the aqueous phase. Cleanup was performed using a RNeasy Total RNA Isolation kit (Qiagen; Valencia, CA). Double-stranded cDNA was synthesized from total RNA using a T7-(dT)24 primer [5'-ggCCAgTgAATTgTAATACgACTCACTATAgggAggCgg-(dT)24-3'] and a Superscript Double-Stranded cDNA Synthesis kit (InVitrogen; Carlsbad, CA). The rat PLM DNA was amplified from the cDNA by PCR using the forward primer 5'-AAgCTTCTCgAgATggCATCTCCCggCCACATCCTg-3', which included restriction sites for HindIII and XhoI at the 5' end, and the reverse primer 5'-AAgCTTAgATCTTTACCgCCTgCgggTggACAgACg-3', which included HindIII and BglII restriction sites for insertion into pAdTrack-CMV. Insertion of the PCR product into pAdTrack-CMV with XhoI at the 5' end and BglII at the 3' end resulted in correct orientation of rASPLM from the CMV promoter. This shuttle vector plasmid was used to construct recombinant Adv expressing both GFP and rASPLM (Adv-GFP-rASPLM) as described above.
Adv infection of cardiac myocytes. Two hours after isolation, myocytes were infected with either Adv-GFP-ASPLM or Adv expressing GFP alone (Adv-GFP) at a multiplicity of infection of 2–5 for 3 h. Media were then changed, and myocytes were studied after 3 days of continuous pacing in culture. We have previously shown that Adv infection of adult rat myocytes was 95% efficient and did not affect myocyte contractility (17, 26). For the sake of brevity, myocytes infected with Adv-GFP, Adv-GFP-dASPLM, and Adv-GFP-rASPLM are referred to as GFP, dASPLM, and rASPLM myocytes, respectively.
Myocyte shortening measurements. Myocytes adherent to the coverslips were bathed in 0.6 ml of air- and temperature-equilibrated (37°C) HEPES-buffered (20 mM, pH 7.4) medium 199 containing either 0.6 or 5.0 mM [Ca2+]o. Measurements of myocyte contraction (1 Hz) were performed as previously described (17, 19, 22, 24, 26).
[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 IM 35 inverted microscope were field stimulated to contract at 1 Hz between platinum wire electrodes (17, 19, 24, 26). [Ca2+]o was either 0.6 or 5.0 mM. [Ca2+]i transient measurements, calibration of fura-2 fluorescent signals, and [Ca2+]i transient analysis were performed as previously described (2, 17, 19, 24, 26).
Caffeine-induced contractures. Fura-2-loaded myocytes bathed in 5.0 mM [Ca2+]o were paced at 1 Hz. At 200 ms after the 21st beat, a trigger TTL pulse (2.4 s) was generated by a programmable multichannel stimulator (STIM-6, Ionoptix; Milton, MA) to initiate caffeine (5 mM) puffer superfusion (19, 23, 24, 26, 27). The [Ca2+]i transient associated with caffeine-induced contracture was captured and analyzed using Ionoptix software.
Na+/Ca2+ exchange current measurements. Whole cell patch-clamp recordings were performed at 30°C as previously described (19, 24, 27). 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. Pipettes were filled with a buffered Ca2+ solution containing (in mM) 100 Cs+ glutamate, 7.25 NaCl, 1 MgCl2, 20 HEPES, 2.5 Na2ATP, 10 EGTA, and 6 CaCl2; pH 7.2. Free Ca2+ in the pipette solution was 205 nM, measured fluorimetrically with fura-2. Myocytes were bathed in an external solution containing (in mM) 130 NaCl, 5 CsCl, 1.2 MgSO4, 1.2 NaH2PO4, 5 CaCl2, 10 HEPES, 10 Na+ HEPES, and 10 glucose; pH 7.4. Verapamil (1 µM) was used to block L-type Ca2+ currents. K+ currents and Na+-K+-ATPase pump currents were minimized by Cs+ substitution for K+ in both pipette and external solutions. Myocytes were selected for electrophysiological studies on the basis of rod-shaped morphology, clear cross-striations, and the absence of membrane blebs. For current measurements, cell capacitance and series resistance were compensated for as best as possible with the analog circuitry of the patch-clamp amplifier. Membrane potential (Em) was held at the calculated reversal potential of Na+/Ca2+ exchanger current (INaCa; –73 mV) for 5 min before stimulation. This precaution minimized fluxes through NCX1 before the voltage ramp and thus allowed cytosolic Na+ concentration ([Na+]i) and [Ca2+]i to equilibrate with those present in the pipette solution. A descending voltage ramp (from +100 to –120 mV; 500 mV/s) was used to prevent activating the voltage-gated Na+ channel. This was immediately followed by an ascending voltage ramp (from –120 to +100 mV; 500 mV/s). The voltage ramp was repeated after the addition of 1 mM CdCl2 to the external solution. Currents were derived from measurements during the descending voltage ramp. INaCa was defined as the difference current measured in the absence and presence of Cd2+. Currents were filtered at 1 kHz, and data were acquired at 2 kHz. Whole cell capacitance for each myocyte was measured by applying a small hyperpolarizing pulse (–10 mV, 16 ms) and integrating the resulting current change (digitized at 50 kHz, 0.5-kHz filter) over time (27). To facilitate comparison of NCX1 currents, INaCa of each myocyte was divided by the whole cell capacitance to account for variations in cell sizes.
PLM, Na+/Ca2+ exchanger, sarco(endo)plasmic reticulum Ca2+-ATPase, calsequestrin, and Na+-K+-ATPase immunoblotting. Cultured myocytes were harvested for immunoblotting on day 3 as previously described (17, 19, 23, 24, 26). Briefly, proteins in myocyte lysates were subjected to 7.5% (NCX1, Na+-K+-ATPase, and calsequestrin) or 12% [PLM and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2)] SDS-PAGE under either nonreducing (10 mM N-ethylmaleimide for NCX1) or reducing (5% 
-mercaptoethanol for PLM, Na+-K+-ATPase, SERCA2, and calsequestrin) conditions. The fractionated proteins were transferred onto Immun-Blot polyvinylidene difluoride (PVDF) membranes. Primary antibodies used were as follows: for PLM, rabbit polyclonal antibodies against the PLM COOH terminus (C2Ab) (1:10,000; Ref. 17); for NCX1, mouse monoclonal antibody (1:1,000, R3F1, Swant; Bellinzona, Switzerland); for SERCA2, mouse monoclonal antibody (1:2,500, MA3–919, Affinity Bioreagents; Golden, CO); for calsequestrin, rabbit anti-calsequestrin antibody (1:2,500, Swant); and for Na+-K+-ATPase, mouse monoclonal antibody (1:250, 
5, Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies used were donkey anti-rabbit and sheep anti-mouse IgG (Amersham). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting system. Protein band signal intensities were quantitated by scanning autoradiograms of the blots with a phosphoimager (Molecular Dynamics; Sunnyvale, CA).
Coimmunoprecipitation experiments. C2Ab was covalently linked to agarose support beads according to the manufacturer's instructions (Affi-Gel Hz immunoaffinity kit, Catalog No. 153-6060, Bio-Rad). Briefly, 1.3 mg of oxidized and desalted C2Ab were added to 1.2 ml of washed Affi-Gel Hz gel and tumbled for 19 h at room temperature. The IgG coupling efficiency was measured to be 94%. After conditioning of the immunoaffinity column, 1.6 ml of myocyte lysate (3.84 mg protein) prepared from freshly isolated adult rat myocytes were mixed with 1.6 ml of PBS and centrifuged at 20,000 g for 10 min at 4°C. The supernatant (2.5 ml, 3 mg protein) was added to the column, and the eluant was collected and reloaded onto the column. This reloading was repeated 50 times. After the column was washed with 0.5 M NaCl followed by PBS, the column was eluted with 0.2 M glycine-HCl (pH 2.5), and 450-µl eluant fractions were collected into tubes containing 50 µl of 1 M Tris base (pH 9.5). The three eluant fractions (500 µl each) with the highest protein concentrations were subjected to 10% SDS-PAGE, followed by protein transfer to PVDF membranes for detection of PLM and NCX1 by Western blotting.
Statistics. All results are expressed as means ± SE. For analysis of a parameter (e.g., systolic [Ca2+]i, maximal contraction amplitude) as functions of group (GFP vs. rASPLM) and [Ca2+]o, two-way ANOVA was used to determine statistical significance. Likewise, two-way ANOVA was used to analyze INaCa as a function of group and membrane voltage. For analyses of the half-time (t1/2) of relaxation from caffeine contracture, whole cell capacitance, and protein abundance, Student's t-test was used. A commercial software package (JMP version 4, SAS Institute; Cary, NC) was used. In all analyses, P < 0.05 was taken to be statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Although the COOH termini (cytoplasmic domain) of rat and dog PLM are identical, there are three amino acid differences in the NH2 termini (extracellular domain) and an additional three amino acid differences in the transmembrane domains between rat and dog PLM (13, 18). We therefore cloned rat heart PLM (METHODS) and used it in the construction of Adv-GFP-rASPLM. At 3 days after Adv infection, PLM protein levels were significantly (P < 0.012) lower in rASPLM compared with control GFP myocytes (275 ± 54 vs. 359 ± 47 arbitrary units, n = 6; Fig. 1). In contrast, neither SERCA2 (539 ± 45 vs. 546 ± 32 arbitrary units, n = 5, P < 0.78), NCX1 (262 ± 27 vs. 238 ± 28 arbitrary units, n = 5, P < 0.14), Na+-K+-ATPase (770 ± 45 vs. 866 ± 146 arbitrary units, n = 4, P < 0.56), nor calsequestrin (400 ± 18 vs. 393 ± 6 arbitrary units, n = 5, P < 0.71) protein levels were different between rASPLM and control GFP myocytes (Fig. 1).
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Effects of PLM downregulation on myocyte contractile function. We have previously shown that compared with control GFP myocytes, PLM overexpression resulted in larger contraction amplitude at 0.6 mM [Ca2+]o but lower contraction amplitude at 5.0 mM [Ca2+]o (17). In the present study, we investigated what effects PLM downregulation would have on myocyte contractility. At 0.6 mM [Ca2+]o, rASPLM myocytes shortened less than GFP myocytes (Fig. 2 and Table 2). In contrast, at 5.0 mM [Ca2+]o, rASPLM myocytes contracted more than GFP myocytes (Fig. 2 and Table 2). These conclusions are supported by highly significant [Ca2+]o (P < 0.0001) and group x [Ca2+]o interaction (P < 0.0003) effects, indicating that the magnitude and/or direction of the effects of [Ca2+]o on cell shortening were different across the experimental groups (rASPLM vs. GFP).
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To further analyze contraction dynamics, we measured maximal shortening and relengthening velocities (Table 2). Maximal shortening and relengthening velocities were significantly lower in rASPLM myocytes at 0.6 mM [Ca2+]o but higher at 5.0 mM [Ca2+]o (group x [Ca2+]o interaction effect, P = 0.0002). In both GFP and rASPLM myocytes, raising [Ca2+]o increased maximal shortening and relengthening velocities (significant [Ca2+]o effect, P < 0.0001).
Effects of PLM downregulation on [Ca2+]i transients. [Ca2+]i occupies a central role in cardiac myocyte excitation-contraction coupling. Therefore, the differences in contractile behavior between GFP and rASPLM myocytes may be related to differences in [Ca2+]i homeostasis brought about by PLM downregulation. Indeed, as a group, diastolic [Ca2+]i was significantly (P < 0.012) lower in rASPLM myocytes (Table 3). Changing [Ca2+]o had no significant effect on diastolic [Ca2+]i (Table 3; [Ca2+]o effect, P = 0.79).
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With respect to systolic [Ca2+]i, at 0.6mM [Ca2+]o, measured values for rASPLM myocytes were lower than those found for GFP myocytes, whereas the reverse was true at 5.0 mM [Ca2+]o (Fig. 3 and Table 3). This conclusion was supported by the results of two-way ANOVA: insignificant group (P = 0.55) but significant [Ca2+]o (P < 0.0001) and group x [Ca2+]o interaction (P < 0.002) effects, indicating that the magnitude and/or direction of the effects of [Ca2+]o on systolic [Ca2+]i were different across GFP and rASPLM myocytes.
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The percent increase in the fura-2 fluorescence intensity ratio is an accurate reflection of [Ca2+]i transient amplitude. Compared with GFP myocytes, the amplitudes of [Ca2+]i transients in rASPLM myocytes were lower at 0.6 mM [Ca2+]o but higher at 5.0 mM [Ca2+]o (Table 3). Two-way ANOVA indicated significant group (P < 0.05), [Ca2+]o (P < 0.0001), and group x [Ca2+]o interaction (P < 0.0001) effects.
The t1/2 of [Ca2+]i decline, an indicator of sarcoplasmic reticulum (SR) Ca2+ uptake activity (23), showed no significant differences between GFP and rASPLM myocytes (Table 3; group effect, P = 0.36; group x [Ca2+]o interaction effect, P = 0.65). Elevating [Ca2+]o, which increased the amplitudes of the [Ca2+]i transient, significantly lowered the t1/2 of [Ca2+]i decline in both GFP and rASPLM myocytes (Table 3; [Ca2+]o effect, P < 0.0001). This observation is consistent with a report by Bers and Berlin (1) showing that the kinetics of [Ca2+]i decline were dependent on peak [Ca2+]i.
Effects of PLM downregulation on INaCa. The pattern of contraction and [Ca2+]i transient changes observed in PLM-downregulated myocytes (Figs. 2 and 3) is similar to that seen in NCX1-overexpressing rat myocytes (26) but opposite to that in which NCX1 was downregulated (19). In addition, we (24) have recently shown that PLM overexpression inhibited INaCa in adult rat myocytes. We therefore investigated whether PLM downregulation would affect INaCa in rat myocytes. Downregulation of PLM did not affect whole cell capacitance [143 ± 7 pF in rASPLM (n = 15) vs. 139 ± 8 pF in GFP (n = 14) myocytes; P < 0.73], indicating that myocyte surface area was unchanged with PLM downregulation. Figure 4B shows membrane currents in a control GFP myocyte subjected to the descending-ascending voltage ramp under conditions in which Ca2+, K+, and Na+-K+-ATPase currents were blocked and in the absence and presence of Cd2+. Note that with the exception of small contamination of the ascending ramp by the cardiac Na+ current, there were little to no differences in currents measured between the descending and ascending voltage ramp. This suggests that [Ca2+]i and [Na+]i sensed by the Na+/Ca2+ exchanger did not appreciably change by NCX1 fluxes during the brief (880 ms) voltage ramp. Figure 4C shows the current-voltage relationship of INaCa density at 30°C and 5.0 mM [Ca2+]o in both GFP (open symbols) and rASPLM (filled symbols) myocytes. The reversal potential of INaCa of approximately –70 mV was similar between GFP and rASPLM myocytes and close to the calculated reversal potential of –73 mV under our experimental conditions. In addition, the absolute magnitudes of INaCa density were, in general, lower in GFP than rASPLM myocytes. Two-way ANOVA confirmed a significant group (GFP vs. rASPLM) effect (P < 0.0013). In both GFP and rASPLM myocytes, depolarization to more positive membrane potentials increased the absolute magnitudes of INaCa (voltage effect, P < 0.0001).
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Effects of PLM downregulation on caffeine-induced contractures. After steady-state twitch amplitude was achieved, application of 5 mM caffeine at end diastole resulted in a large [Ca2+]i increase due to SR Ca2+ release (Fig. 5). In the continued presence of caffeine, decline of [Ca2+]i is known to be mediated primarily by forward Na+/Ca2+ exchange, because SR Ca2+ accumulation is inhibited (27). At 5 mM [Ca2+]o, the t1/2 of [Ca2+]i decline after caffeine-induced SR Ca2+ release was significantly (P < 0.03) faster in rASPLM (1.89 ± 0.21 s, n = 10; Fig. 5B) than GFP (2.62 ± 0.22 s, n = 10; Fig. 5A) myocytes, indicating enhanced forward Na+/Ca2+ exchange in rASPLM myocytes.
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Association of PLM with NCX1 in adult rat myocytes. The results of INaCa and caffeine-induced Ca2+ release experiments suggest that PLM may directly interact with NCX1. In addition, we (24) have previously demonstrated colocalization of PLM with NCX1 at the sarcolemma, t tubules, and intercalated disks in adult rat myocytes. We therefore sought to examine the physical association between PLM and NCX1. Figure 6 demonstrates that the PLM antibody, but not preimmune serum, when covalently linked to agarose support beads, was able to precipitate both PLM and NCX1 present in native adult rat myocyte lysates.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Our initial attempt to downregulate PLM in adult rat myocytes using an AS full-length construct based on the dog PLM coding sequence was unsuccessful in that neither PLM protein levels (RESULTS) nor myocyte contractility amplitudes (Table 1) were affected by Adv-GFP-dASPLM exposure. This fortuitous result provides a very useful control for our successful experiments using rASPLM in that similar lengths of DNA sequence were employed in rASPLM and dASPLM construction and indicates downregulation of PLM required species-specific AS constructs. Using Adv-mediated rASPLM delivery, we were able to downregulate PLM protein in adult rat myocytes by 23% after 3 days (Fig. 1). It is important to note that expression of other important membrane ion transport proteins such as NCX1, Na+-K+-ATPase, and SERCA2 was not affected by exposure to rASPLM (Fig. 1), indicating the absence of global suppression of translation that may be associated with long DNA sequences used in AS delivery. In addition, SR Ca2+ uptake, as estimated by the t1/2 of [Ca2+]i transient decline (20, 23), was unaffected (Table 2). These observations suggested that AS PLM downregulation was specific.
Downregulation of PLM protein by Adv-mediated AS delivery was accompanied by alterations in myocyte contractility (Fig. 2 and Table 2). Specifically, compared with control GFP myocytes, maximal contraction amplitude and maximal shortening and relengthening velocities in rASPLM myocytes were significantly lower at 0.6 mM but higher at 5.0 mM [Ca2+]o. The pattern of contractile abnormalities with PLM downregulation was exactly opposite to that observed in PLM-overexpressing myocytes (17). The cumulative evidence strongly suggests that one of the physiological functions of PLM in cardiac tissues is regulation of cardiac contractility.
The mechanism by which PLM regulates cardiac contractility is most likely mediated by modulating Ca2+ fluxes. In rat cardiac myocytes in which PLM was overexpressed, [Ca2+]i transient amplitudes were higher at 0.6 mM [Ca2+]o but lower at 5 mM [Ca2+]o (17). In contrast, PLM downregulation resulted in [Ca2+]i transient amplitudes that were lower at 0.6 mM [Ca2+]o but higher at 5 mM [Ca2+]o (Fig. 3 and Table 3). Note that the pattern of [Ca2+]i transient amplitude alterations by increasing or decreasing PLM in rat cardiac myocytes mirrored the pattern of contractile behavior differences, supporting our hypothesis that PLM modulated cardiac contractility by perturbing Ca2+ fluxes.
Of the major Ca2+ transporters in cardiac sarcolemma, only the Na+/Ca2+ exchanger can mediate both Ca2+ influx (reverse NCX1 mode) and efflux (forward NCX1 mode), depending on the thermodynamic driving force. We (24) have previously demonstrated that PLM overexpression directly inhibited NCX1 activity in adult rat cardiac myocytes. Our present observations on the effects of PLM downregulation on myocyte contractility (Fig. 2 and Table 2) and [Ca2+]i transients (Fig. 3 and Table 3) can be explained by relief of NCX1 inhibition associated with PLM downregulation. The lower diastolic [Ca2+]i values observed in rASPLM myocytes (Table 3) supports this concept and is consistent with the notion that because forward NCX1 was necessary in maintaining diastolic [Ca2+]i levels (20), increased forward NCX1 activity in rASPLM myocytes resulted in lower diastolic [Ca2+]i levels. In this regard, diastolic [Ca2+]i levels were also significantly lower in adult rat myocytes in which NCX1 was overexpressed (26). Another piece of evidence supporting enhanced NCX1 activity in PLM-downregulated myocytes is the faster rate of relaxation from caffeine-induced contracture in rASPLM myocytes (Fig. 5), indicating accelerated forward NCX1 activity. In addition, INaCa in rASPLM myocytes was significantly higher in rASPLM myocytes (Fig. 4), indicating enhanced NCX1 activity. Note that the conditions used in our INaCa measurements were carefully designed to minimize contamination by other currents such as L-type Ca2+ currents, Na+-K+-ATPase currents, and Na+ and K+ currents. In addition, the thermodynamic parameters ([Na+]i, [Ca2+]i, [Na+]o, and [Ca2+]o) that determined the equilibrium potential (ENaCa) of INaCa, and hence its driving force (Em – ENaCa), were identical between control and rASPLM myocytes. Thus the differences in INaCa between GFP and rASPLM myocytes could be unambiguously assigned to the effects of PLM downregulation. Finally, the major finding that PLM coimmunoprecipitates with NCX1 in native adult rat myocytes (Fig. 6) strongly indicates that PLM directly interacted with NCX1 and is consistent with our previous finding that PLM and NCX1 colocalized at the sarcolemma, t tubules, and intercalated disks of rat cardiac myocytes (24).
Regulation of NCX1 activity by PLM could explain the differential effects of low versus high [Ca2+]o on [Ca2+]i transient and contraction amplitudes in PLM-overexpressing (17) and -downregulated myocytes (Tables 2 and 3), as suggested by the following model. During diastole, the primary function of NCX1 is to extrude Ca2+ (20). This is consistent with our observations that overexpressing (26) or downregulating (19) NCX1 resulted in lower or higher diastolic [Ca2+]i levels, respectively. Upon depolarization, when Em exceeds ENaCa, Ca2+ influx is thermodynamically favored, although estimates of the amount and duration of Ca2+ influx via reverse NCX1 during systole are at present imprecise. At high [Ca2+]o (5 mM), by increasing NCX1 amounts (with overexpression) or its activity (by PLM downregulation) without affecting other Ca2+ transport pathways (19, 26), more Ca2+ would initially enter via reverse NCX1 during systole. Increased Ca2+ influx resulted in the observed higher SR Ca2+ content and larger [Ca2+]i transient and contraction amplitudes in myocytes overexpressing NCX1 compared with control GFP myocytes (26). The higher SR Ca2+ content led to a corresponding increase in Ca2+ spark frequency, resulting in increased SR Ca2+ leak, which limited the otherwise inexorable increase in SR Ca2+ content. The increased SR Ca2+ leak, together with the Ca2+ that entered via L-type Ca2+ channels, was extruded by NCX1 during diastole. In this way, the NCX1-overexpressing myocyte stimulated at 5 mM [Ca2+]o reached a higher steady-state SR Ca2+ (compared with the control GFP myocyte) and maintained beat-to-beat Ca2+ balance. At low [Ca2+]o (0.6 mM), the driving force for Ca2+ influx was much reduced during systole, but increased NCX1 amounts (by overexpression) or its activity (by PLM downregulation) would initially pump more Ca2+ out during diastole (compared with the control GFP myocytes), thus partially depleting SR Ca2+ content until a new lower steady-state SR Ca2+ was reached. Indeed, compared with control GFP myocytes, at 0.6 mM [Ca2+]o, diastolic [Ca2+]i was lower and SR Ca2+ content and [Ca2+]i transient and contraction amplitudes were all reduced in NCX1-overexpressing myocytes (26). The same paradigm can be successfully applied to explain our observations on NCX1-downregulated (19) or PLM-overexpressing (NCX1 inhibited) myocytes (17). It is the unique ability of NCX1 to drive Ca2+ in and pump Ca2+ out during an action potential that explains the differential effects of low versus high [Ca2+]o on [Ca2+]i transient and contraction amplitudes in myocytes in which NCX1 amounts or activities were changed.
PLM has also been shown to associate with Na+-K+-ATPase and inhibits its activity (4, 25). Relief of inhibition of Na+-K+-ATPase by PLM downregulation would theoretically lower [Na+]i and result in indirect modulation of NCX1 activity and thereby affect contractility. This mechanism is unlikely to explain our observations in rASPLM myocytes. First, increased Na+-K+-ATPase activity associated with PLM knockdown would lower [Na+]i, resulting in increased forward Na+/Ca2+ exchange but decreased reverse Na+/Ca2+ exchange. This would result in decreased contraction amplitudes in rASPLM myocytes studies under both low and high [Ca2+]o conditions, a prediction not consistent with our observations in rASPLM myocytes (Fig. 2 and Table 2). Second, in our whole cell patch-clamp experiments in which Na+-K+-ATPase activity was minimized by the absence of K+ in both intracellular and extracellular solutions, and under conditions in which the driving force (Em – ENaCa) for INaCa was identical between control and rASPLM myocytes, INaCa was still higher in rASPLM myocytes (Fig. 4).
Comparing myocytes isolated from rat hearts 3–8 wk after MI with those isolated from sham hearts, contraction amplitudes were higher at 0.6 mM [Ca2+]o and lower at 5.0 mM [Ca2+]o (21–23), opposite to what was observed in rASPLM myocytes (Fig. 2 and Table 2). In addition, both INaCa (27) and Na+-dependent Ca2+ uptake in sarcolemmal vesicles (5) were depressed and relaxation from caffeine-induced contracture was prolonged (22) in MI myocytes; again opposite to what was observed in rASPLM myocytes (Figs. 4 and 5). Interestingly, PLM expression was increased in rat hearts post-MI (15). Because PLM overexpression inhibited NCX1 activity (26) and simulated contractile abnormality post-MI (17, 22), an intriguing therapeutic modality is to downregulate PLM post-MI by PLM AS delivery. The results of our present study demonstrated the feasibility of this approach.
There are some limitations to the present study. The first is that we did not measure [Ca2+]i transient and contraction amplitudes in control GFP and rASPLM myocytes incubated at an intermediate [Ca2+]o of 1.8 mM. Previous studies in adult rat myocytes indicated that [Ca2+]i transient and contraction amplitudes measured at 1.8 mM [Ca2+]o were not affected by MI (22) or by overexpressing (26) or downregulating NCX1 (19). In addition, the effects of PLM overexpression on [Ca2+]i transient and contraction amplitudes, which were at least partly mediated via the inhibitory effect of PLM on NCX1 (24), were much more apparent at 0.6 and 5.0 than at 1.8 mM [Ca2+]o (17). We therefore reasoned that [Ca2+]i transient and contractility changes associated with PLM downregulation would be sufficiently characterized with measurements at 0.6 and 5.0 mM [Ca2+]o. Another limitation is that we ignored the observations in noncardiac tissues showing that PLM forms channels that are permeable to the zwitterion taurine (10) and participates in regulatory volume decrease of cells (11, 12). Whole cell capacitance, however, was not changed in myocytes in which PLM was overexpressed (24) or downregulated (RESULTS). This observation suggests that steady-state myocyte membrane surface area, and by extrapolation cell volume, was not affected by PLM overexpression or downregulation. Whether changing PLM levels will affect the ability of adult myocytes to respond to quick changes in osmolarity (e.g., regulatory volume decrease) is beyond the scope of the present study but worthy of future investigation.
In summary, we demonstrated that downregulation of PLM by Adv-mediated AS delivery resulted in altered patterns of contraction and [Ca2+]i transients, lower diastolic [Ca2+]i levels, enhanced INaCa, and faster relaxation from caffeine-induced contractures. Expression of SERCA2, NCX1, Na+-K+-ATPase, and calsequestrin, as well as SR Ca2+ uptake, were unaffected by PLM downregulation. We conclude that the most consistent explanation of our observations is that PLM downregulation decreased its inhibition of NCX1 activity, leading to increases in both Ca2+ influx and efflux during a twitch, with resultant altered contractile behavior. In addition, we demonstrated an association of PLM and NCX1 in native adult rat cardiac myocytes. Because PLM was overexpressed in post-MI myocytes, and because the pattern of contractile abnormalities in PLM-overexpressing myocytes mimicked that observed in post-MI myocytes, we speculate that downregulation of PLM by AS treatment may offer a rational therapeutic approach postinfarction.
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported in part by National Institutes of Health Grants HL-58672 (to J. Y. Cheung), DK-46678 (to J.Y. Cheung, coinvestigator), GM-46691 (to L. I. Rothblum), HL-70548 and GM-64640 (to J. R. Moorman), and HL-69074 (A.L. Tucker); by American Heart Association Pennsylvania Affiliate Grants-In-Aid 0265426U (to X. Zhang) and 0355744 U (to J. Y. Cheung); and by grants from the Geisinger Foundation (to J. Y. Cheung and L. I. Rothblum).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. A. Mirza and X.-Q. Zhang contributed equally to this study. 
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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; n = 7) and rASPLM (
, n = 6) myocytes. The reversal potential of INaCa in both GFP and rASPLM myocytes was approximately –70 mV. Note the enhancement of INaCa in myocytes in which PLM was downregulated. Error bars are not shown if they fall within the boundaries of the symbol.
