Regulation of cardiac myocyte contractility by phospholemman: Na⁺/Ca²⁺ exchange versus Na⁺-K⁺-ATPase

METHODS

Generation of PLM-deficient mice and animal care.

PLM KO mice backcrossed to a pure congenic C57BL/6 background were generated as previously described (12, 22). Homozygous adult littermates of ∼3 mo old were used. Mice were housed and fed on a 12:12-h light-dark cycle at the Thomas Jefferson University Animal Facility and were supervised by veterinary staff members. Standard care was provided to all mice used for experiments. All protocols applied to the mice in this study were approved and supervised by the Institutional Animal Care and Use Committees at Thomas Jefferson University and the University of Virginia.

Isolation and culture of adult murine cardiac myocytes.

Cardiac myocytes were isolated from the septum and left ventricular free wall of wild-type (WT) and PLM KO mice (∼3 mo old, 25–37g) according to the protocol of Zhou et al. (30) and as modified by us (22). Isolated myocytes were plated on laminin-coated coverslips, and the Ca ^{concentration of the buffer was progressively increased from 0.05 to 0.125 to 0.25 to 0.5 mM (10-min intervals each). The 0.5 mM Ca buffer was then aspirated and replaced with minimal essential medium (MEM) containing 1.2 mM Ca, 2.5% FBS, and antibiotics (1% penicillin-streptomycin). The pH was adjusted to 7.0 in 4% CO}₂ by the addition of NaHCO₃ (0.57 g/l). After 1 h (4% CO₂, 37°C), the medium was replaced with FBS-free MEM containing 0.1 mg/ml BSA, antibiotics, and insulin-transferrin-selenium (ITS) supplement. 2,3-Butanedione monoxime (BDM; 10 mM) was present throughout the heart perfusion and culture. Media were changed every day. Before measurements of contraction, [Ca^]_i transients, I_NaCa, I_pump, and sarcoplasmic reticulum (SR) Ca ^{content, culture medium containing BDM was aspirated, and cells were bathed with MEM without BDM and returned to the incubator (37°C) for 30 min. Coverslips containing cultured myocytes were then taken out of the incubator, mounted in the Dvorak-Stotler chamber, and bathed in fresh media before measurements.}

Staining of t-tubules in myocytes.

Myocytes isolated from WT hearts and cultured for 0, 24, and 48 h were stained with di-8-ANEPPS (10 μM) at 37°C for 10 min. Stained myocytes were washed with MEM followed by examination using both confocal (Zeiss LSM510, excitation and emission wavelengths at 488 and 505 nm, respectively) and conventional wide-field fluorescence microscopy (Olympus IX71, excitation and emission wavelengths at 480 ± 20 and 535 ± 25 nm, respectively). In some experiments, myocytes were exposed to formamide (1.5 mol/l) at 37°C for 15 min to induce detubulation before being imaged.

Adenoviral infection of cardiac myocytes.

Recombinant, replication-deficient adenovirus (Adv) expressing either green fluorescent protein (GFP) alone, GFP and WT dog PLM, or GFP and dog PLM mutants (S68A or S68E) were constructed as previously described (17, 18). Two hours after isolation, myocytes were infected with Adv-GFP, Adv-GFP-PLM, or Adv-GFP-PLM mutants at a multiplicity of infection of 2 for 3.5 h. Media were then changed, and myocytes were studied after 48 h. For the sake of brevity, PLM KO myocytes infected with Adv-GFP, Adv-GFP-PLM, and Adv-GFP-PLM mutants are referred to as KO-GFP, KO-PLM, and the respective designation for the PLM mutant (e.g., KO-S68A) myocytes, respectively.

Myocyte shortening measurements.

Myocytes adherent to 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 1.8 or 5.0 mM [Ca^]_o. Measurements of myocyte contraction (1 Hz) were performed as previously described (14, 17, 18, 21, 22, 29).

[Ca^]_i transient measurements.

Myocytes loaded with fura-2 (0.67 μM, 15 min, 37°C) were field stimulated to contract (1 Hz, 37°C) in medium 199 containing either 1.8 or 5.0 mM [Ca^]_o. [Ca^]_i transient measurements, daily calibration of fura-2 fluorescent signals, and [Ca^]_i transient analyses were performed as previously described (14, 17, 18, 21, 22, 29).

I_NaCa, I_pump, and SR Ca ^{content measurements.}

Whole cell patch-clamp recordings were performed at 30°C as previously described (21, 22). The pipette diameter was 4–6 μm and the pipette resistance was 0.8–1.4 MΩ when filled with standard internal solution. For I_NaCa measurements, the pipette solution contained (in mM) 100 Cs^{-glutamate, 7.25 Na-HEPES, 1 MgCl}₂, 12.75 HEPES, 2.5 Na₂ATP, 10 EGTA, and 6 CaCl₂ (pH 7.2). Free Ca ^{in the pipette solution was 205 nM. Myocytes were bathed in an external solution containing (in mM) 130 NaCl, 5 CsCl, 1.2 MgSO}₄, 1.2 NaH₂PO₄, 5 CaCl₂, 10 HEPES, 10 Na^{-HEPES, and 10 glucose (pH 7.4) (}14, 18, 25). Verapamil (1 μM), ouabain (1 mM), and niflumic acid (10 μM) were used to block L-type Ca ^currents, I_pump, and Cl ^{currents, respectively. The myocyte was held at the calculated equilibrium potential for} I_NaCa (E_NaCa) of −73 mV for at least 5 min before current was elicited with a descending-ascending voltage ramp (from +100 to −120 and back to +100 mV, 500 mV/s). I_NaCa was defined as the difference current in the absence and presence of CdCl₂ (1 mM). Our conditions for measuring I_NaCa were carefully chosen to minimize contamination by Na^{-K-ATPase activity (K free and in the presence of ouabain) and ion fluxes through NCX1 before the onset of voltage ramp (by holding the cell at the calculated} E_NaCa), thereby allowing [Na^]_i and [Ca^]_i to equilibrate with those present in the pipette solution.

For I_pump measurements (26), the standard pipette solution contained (in mM) 70 Na^{-aspartate, 20 K-aspartate, 8 CsOH, 7 MgSO}₄, 11 EGTA, 10 TEA.Cl, 1 CaCl₂, 5 HEPES, 5 Na₂ATP, and 0.2 GTP (pH 7.2). To decrease the pipette Na ^{concentration ([Na]}_pip) to 10 mM, Cs ^{was substituted for Na. The external solution contained (in mM) 137.7 NaCl, 18 KCl, 2.3 NaOH, 1 MgCl}₂, 2 BaCl₂, 1 CdCl₂, 5 HEPES, and 10 glucose (pH 7.4). The holding potential was switched from −70 to −40 mV (300 ms) before the application of a negative voltage ramp (from +60 to −120 mV, 20 mV/s), first in the absence and then in the presence of 1 mM ouabain. I_pump was defined as the difference current before and after the addition of ouabain.

SR Ca ^{content was estimated by integrating forward} I_NaCa induced by caffeine exposure as previously described (21, 22, 29). The pipette solution consisted of (in mM) 100 Cs^{-glutamate, 1 MgCl}₂, 30 HEPES, and 2.5 MgATP (pH 7.2). The external solution contained (in mM) 130 NaCl, 5 CsCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 CaCl₂, 20 HEPES, and 10 glucose (pH 7.4, 30°C). The holding potential was −70 mV. At 200 ms after the 11th conditioning pulse (from −70 to 0 mV, 300 ms, 1 Hz), with membrane potential held at −70 mV, caffeine (5 mM, 2.4 s) was applied by puffer superfusion. The resulting inward current was digitized at 0.5 kHz and collected for 5 s. To convert the I_NaCa time integral (in Coulombs) to moles, the charge was divided by Faraday's constant of 96,487 coulombs/equivalent, based on 3 Na ^{being exchanged for each Ca. SR Ca content was normalized to cell size (in fmol/fF).}

Immunoblot analysis.

Myocytes were harvested for immunoblot analysis after 48 h of culture as previously described (14, 17, 18, 21, 26). For PLM immunoblot analysis, proteins in myocyte lysates were subjected to 12% SDS-PAGE under reducing (5% β-mercaptoethanol) conditions. Polyclonal C2 antibody (1:10,000) was used to detect the COOH-termini of both endogenous mouse and WT dog PLM (the predominantly unphosphorylated form) (16, 17, 26). Monoclonal B8 antibody (1:5,000) was used to detect the NH₂-termini of WT dog PLM and its Ser ^{mutants (}18). Polyclonal CP68 antibody (1:500) was used to detect PLM phosphorylated at Ser ⁽16, 26).

For the detection of NCX1, sarco(endo)plasmic reticulum Ca^{-ATPase 2 (SERCA2), calsequestrin, and the α}₁-subunit of Na^{-K-ATPase, myocyte homogenates were subjected to 7.5% SDS-PAGE under nonreducing (10 mM} N-ethylmaleimide for NCX1) or reducing (for SERCA2, calsequestrin, and Na^{-K-ATPase) conditions. The primary antibodies used were as follows: for NCX1, π11-13 polyclonal antibody (1:700, Swant, Bellinzona, Switzerland); for SERCA2, BL337 polyclonal antibody (1:1,000, Bethyl Laboratories, Montgomery, TX); for calsequestrin, rabbit anti-calsequestrin antibody (1:5,000, Swant); and for the α}₁-subunit of Na^{-K-ATPase (1:1,000, Upstate, Charlottesville, VA). The secondary antibodies used were donkey anti-rabbit and sheep anti-mouse IgG (Amersham). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blot system. Protein band signal intensities were quantitated by scanning autoradiograms of the blots with a phosphorimager.}

Statistics.

All results are expressed as means ± SE. For the analysis of a parameter (e.g., maximal contraction amplitude) as a function of days in culture and [Ca^]_o, two-way ANOVA was used. When the parameter was examined as a function of group (e.g., WT vs. PLM KO), [Ca^]_o, and days in culture, three-way ANOVA was used. For the analysis of ion currents as a function of group and voltage or as a function of group and [Na^]_pip, two-way ANOVA was used. A commercial software package (JMP version 7, SAS Institute, Cary, NC) was used. In all analyses, P < 0.05 was taken to be statistically significant.

RESULTS

Culture and adenoviral infection of adult mouse myocytes.

In our hands, adult mouse myocytes survived better in culture when plated on laminin- rather than Matrigel-coated coverslips. Cultured mouse myocytes maintained their elongated rod shapes when cultured in FBS for 48 h. With serum-free culture, the inclusion of BDM, ITS supplement, and BSA in MEM gave the best results in terms of the maintenance of rod shapes (∼65%) after 48 h (Fig. 1). When infected with adenovirus expressing GFP, green fluorescence started to appear by 18–24 h and increased throughout 48 h of culture (Fig. 1). With few exceptions, GFP fluorescence was much more intense in rod-shaped myocytes compared with rounded and hypercontracted myocytes. Other agents that inhibited myocyte contraction [blebbistatin (5.7 μM) or cytochalasin D (2 μM)] were not as effective as BDM in terms of maintaining myocyte viability in culture. The required presence of BDM precluded pacing adult mouse myocytes while in culture.

Fig. 1.

Culture and adenoviral infection of adult mouse myocytes. Cardiac myocytes were isolated from adult congenic phospholemman (PLM) knockout (KO) mice, infected with adenovirus expressing both green fluorescent protein (GFP) and the PLMS68A mutant, and placed in culture as described in methods. A: transmitted light image of adult mouse myocytes after 24 h of culture. B: fluorescent image of A demonstrating the expression of GFP. C: transmitted light image of another adult mouse myocyte culture at 48 h. D: fluorescent image of C. Both B and D are raw images and were taken at the same time using identical excitation light intensities, with the camera set at the same gain but with autoexposure mode. The higher background in B compared with D is due to the longer exposure time because of the much lower GFP fluorescence emission from adenovirus-infected myocytes cultured for 24 h. In contrast, GFP fluorescence emission from infected myocytes cultured for 48 h was much stronger and required a shorter exposure time, as indicated by the lower background.

Effects of culture and adenoviral infection on whole cell membrane capacitance, t-tubules, and selected proteins involved in excitation-contraction coupling.

Membrane capacitance (C_m) in PLM KO myocytes cultured for 48 h (180 ± 5 pF, n = 27) was not significantly (P < 0.065) different than that measured in freshly isolated PLM KO myocytes (164 ± 5 pF, n = 50), suggesting that myocyte surface membrane area did not appreciably change with short-term culture. When di-8-ANEPPS was used to stain surface sarcolemma and t-tubules, myocytes cultured for 24 and 48 h retained their t-tubular network compared with freshly isolated myocytes (Fig. 2). Pretreatment with formamide resulted in a complete loss of t-tubules (not shown).

Fig. 2.

Effects of culture on t-tubules in mouse cardiac myocytes. Cardiac myocytes were isolated from wild-type (WT) C57BL/6 mice, plated on laminin-coated coverslips, and cultured for 24–48 h. Myocytes were stained with di-8-ANEPPS and imaged with both conventional wide-field fluorescence (A–F) and confocal (G–I) microscopes. Images acquired with ×10 (A–C), ×60 (D–F), and ×100 (G–I) objectives of myocytes at day 0 (A, D, and G), day 1 (B, E, and H), and day 2 (C, F, and I) of culture are shown.

There were no significant differences in the protein levels of NCX1, the α₁-subunit of Na^{-K-ATPase, SERCA2, and calsequestrin between freshly isolated WT and PLM KO myocytes (}Fig. 3 and Table 1), in agreement with our previous results (22) and those of others (2). After 48 h of culture, PLM KO myocytes retained similar amounts of NCX1 and SERCA2 but suffered an ∼18% loss in the α₁-subunit of Na^{-K-ATPase compared with freshly isolated PLM KO myocytes (}Fig. 3 and Table 1). Infection of PLM KO myocytes with adenovirus expressing either GFP or WT PLM followed by culture for 48 h did not have any appreciable effects on the protein levels of NCX1 and SERCA2 but resulted in a similar reduction in the α₁-subunit of Na^{-K-ATPase compared with freshly isolated myocytes (}Fig. 3 and Table 1).

Fig. 3.

Effects of culture on selected proteins involved in excitation-contraction coupling. Myocyte homogenates were prepared from day 0 WT (WT-D0), day 0 PLM KO (KO-D0), day 2 PLM KO (KO-D2), day 2 GFP-expressing KO (KO-GFP), and day 2 PLM-expressing KO (KO-PLM) myocytes and subjected to SDS-PAGE followed by Western blot analysis. Primary antibodies (methods) were used to detect cardiac Na^{/Ca exchanger (NCX1), the α}₁-subunit of Na^{-K-ATPase, sarco(endo)plasmic reticulum Ca-ATPase 2 (SERCA2), and calsequestrin (CLSQ). Composite results are shown in} Table 1.

Table 1.

Effects of culture and adenoviral infection on selected proteins in PLM KO myocytes

	WT-D0	KO-D0	KO-D2	KO-GFP	KO-PLM
Na/Ca exchanger isoform 1	5.2±0.7	5.3±0.5	4.8±0.2	5.6±0.6	5.9±0.2
Sarco(endo)plasmic reticulum Ca-ATPase 2	9.0±0.6	8.9±0.2	8.2±1.1	7.9±0.2	8.0±0.3
α1-Subunit of Na-K-ATPase	9.9±0.5	8.1±1.0	6.6±0.2*	6.4±0.2*	5.5±1.0*

Values are means ± SE (in arbitrary units). WT-D0, day 0 wild-type (WT) myoctyes; KO-D0, day 0 phospholemman (PLM) knockout (KO) myocytes; KO-D2, day 2 PLM KO myocytes; KO-GFP, PLM KO myocytes infected with adenovirus expressing green fluorescent protein (GFP); KO-PLM, PLM KO myocytes infected with adenovirus expressing PLM + GFP. Except for the WT-D0 group (n = 3), all other groups consisted of myocytes prepared from 4 different mice. To correct for slight variations in protein loading, the protein band intensities shown in Fig. 3 were normalized to the corresponding calsequestrin levels.

^P < 0.001 compared with WT-D0 myocytes.

PLM KO myocytes, when infected with adenovirus expressing either WT PLM or its Ser ^{mutants, expressed the constructs robustly after 48 h of culture (}Fig. 4). WT PLM, either endogenous in WT-GFP myocytes or exogenous in KO-PLM myocytes, was partially phosphorylated, as detected by the phospho-specific CP68 antibody (Fig. 4). In addition, protein levels of both phosphorylated and unphosphorylated exogenous PLM in KO-PLM myocytes were much higher than those present in WT-GFP myocytes.

Fig. 4.

Expression of WT PLM and its Ser ^{mutants in cultured PLM KO myocytes. Adult cardiac myocytes isolated from congenic PLM KO mice were infected with adenovirus expressing either GFP alone (KO-GFP), GFP + WT dog PLM (KO-PLM), GFP + PLMS68A (KO-S68A), or GFP + PLMS68E (KO-S68E) and cultured for 48 h. Myocytes from WT littermates infected with adenovirus expressing GFP (WT-GFP) and cultured for 48 h were used as controls. Proteins in myocyte lysates (50 μg/lane) were separated by gel electrophoresis and transferred to ImmunBlot polyvinylidene difluoride membranes. Endogenous mouse and WT dog PLM, unphosphorylated and phosphorylated at Ser, was probed with C2 and CP68 anti-PLM antibodies, respectively, whereas dog PLM Ser mutants were detected with B8 anti-PLM antibody. C2 antibody recognizes predominantly the unphosphorylated COOH-terminus of both dog and rodent PLM, and CP68 antibody is specific for PLM phosphorylated at Ser (}16, 26), whereas B8 antibody recognizes the NH₂-terminus of dog but not rodent PLM (18).

Rescue of PLM deficiency in PLM KO myocytes.

Since PLM KO myocytes maintained their contractile phenotype after short-term culture (Table 2), we next determined if the contractile phenotype would revert back to that observed for WT myocytes after “rescue” with PLM. Compared with KO-GFP myocytes, contraction amplitudes in KO-PLM myocytes were similar at 1.8 mM [Ca^]_o but lower at 5.0 mM [Ca^]_o (Fig. 5 and Table 4; group × [Ca^]_o interaction effect, P < 0.025). There were no differences in contraction amplitudes between WT and KO-PLM myocytes, indicating that PLM rescue was successful in restoring the contractile phenotype in PLM KO myocytes back to that observed in WT myocytes. [Ca^]_i transients in PLM KO myocytes were similarly affected by PLM overexpression: systolic [Ca^]_i and [Ca^]_i transient amplitudes were similar at 1.8 mM [Ca^]_o but significantly lower at 5.0 mM [Ca^]_o (Fig. 5 and Table 5). The half-life (t_1/2) of [Ca^]_i decline, an estimate of in situ SR Ca ^{uptake activity (}27), was not different between KO-PLM and KO-GFP myocytes (Table 5), indicating that PLM had no effect on SERCA2 activity. In support of this interpretation, there were no differences in SERCA2 protein levels between KO-GFP and KO-PLM myocytes (Fig. 3 and Table 1). Importantly, both I_NaCa (Fig. 6) and I_pump (Fig. 7) were significantly suppressed in KO-PLM myocytes compared with KO-GFP myocytes. Compared with WT myocytes, both I_NaCa (Fig. 6) and I_pump (Fig. 7) were higher in KO-GFP but much lower in KO-PLM myocytes. Severe suppression of I_NaCa and I_pump in KO-PLM myocytes is likely due to the supraphysiological levels of both phosphorylated and unphosphorylated exogenous PLM in KO-PLM myocytes compared with the endogenous levels of PLM found in WT myocytes (Fig. 4). Despite no differences in SR Ca ^{uptake activity and SERCA2 protein levels, SR Ca content measured at 5 mM [Ca]}_o was higher in KO-GFP compared with KO-PLM myocytes (Table 6).

Fig. 5.

Effects of PLM and its Ser ^{mutants on contractility and cytosolic Ca concentration ([Ca]}_i) transients in cultured PLM KO myocytes. Adult cardiac myocytes isolated from congenic PLM KO myocytes were infected with adenovirus expressing either GFP alone (A and E), GFP + WT dog PLM (B and F), GFP + PLMS68A (C and G), or GFP + PLMS68E (D and H) and cultured for 48 h. Myocytes were incubated at 5 mM extracellular Ca ^{concentration at 37°C and paced to contract at 1 Hz. Steady-state myocyte contractions (}A–D) and [Ca^]_i transients (E–H) are shown. Composite data are shown in Tables 4–6.

Fig. 6.

Effects of WT PLM and its Ser ^{mutants on Na/Ca exchange current (}I_NaCa) in cultured PLM KO myocytes. Adult cardiac myocytes from congenic PLM KO mice were infected with adenovirus expressing either GFP alone, GFP + WT PLM, GFP + PLMS68A, or GFP + PLMS68E. After myocytes had been cultured for 48 h, I_NaCa was measured as described in methods. Current density-voltage relationships of I_NaCa (means ± SE) from KO-GFP (□; n = 11), KO-PLM (▴; n = 11), KO-S68A (•; n = 7), and KO-S68E (○; n = 9) myocytes are shown. For comparison, current density-voltage relationships of I_NaCa from WT (⧫; n = 20) myocytes are also shown. The reversal potential of I_NaCa in all myocytes examined was approximately −60 mV. Note that I_NaCa was lower in WT compared with KO-GFP myocytes. In addition, I_NaCa was suppressed by WT PLM and the PLMS68E mutant but not by the PLMS68A mutant. Error bars are not shown if they fall within the boundaries of the symbol.

Fig. 7.

Effects of WT PLM and its Ser ^{mutants on Na-K-ATPase current (}I_pump) in cultured PLM KO myocytes. Adult myocytes from congenic PLM KO hearts were infected with adenovirus expressing either GFP alone, GFP + WT PLM (PLM), GFP + PLMS68A (S68A), or GFP + PLMS68E (S68E). After 48 h of culture, I_pump was measured at 18 mM extracellular K ^{concentration at 0 mV at 30°C with pipette Na concentrations ([Na]}_pip) of either 10 mM (open bars) or 80 mM (solid bars). For comparison, I_pump of WT myocytes is shown. At 10 mM [Na^]_pip, there were 8 WT, 9 KO-GFP, 10 KO-PLM, 6 KO-S68A, and 7 KO-S68E myocytes. At 80 mM [Na^]_pip, there were 5 WT, 7 KO-GFP, 6 KO-PLM, 8 KO-S68A, and 8 KO-S68E myocytes. Two-way ANOVA indicated significant differences between WT and KO-GFP (P ≤ 0.05), WT and KO-PLM (P < 0.0004), WT and KO-S68A (P < 0.001), KO-GFP and KO-PLM (P < 0.002), KO-GFP and KO-S68A (P < 0.003), KO-PLM and KO-S68E (P < 0.003), and KO-S68A and KO-S68E (P < 0.004) myocytes, but not between KO-GFP and KO-S68E (P < 0.8), KO-PLM and KO-S68A (P < 0.8), and WT and KO-S68E (P < 0.3) myocytes.

Table 4.

Reversion to the WT contractile phenotype in PLM KO myocytes infected with advenovirus containing PLM

	Day 0		Day 1		Day 2
	Means ± SE	n	Means ± SE	n	Means ± SE	n
1.8 mM [Ca]o
WT	5.28±0.44	16	5.25±0.42	14	5.08±0.48	7
KO-GFP			5.34±0.22	14	4.91±0.33	16
KO-PLM			5.39±0.34	9	5.16±0.40	4
5.0 mM [Ca]o
WT	8.77±0.34	15	8.06±0.41	19	7.59±0.48	15
KO-GFP			9.18±0.55	21	8.99±0.22	26
KO-PLM			7.97±0.48*	18	7.35±0.33*	13

Values are means ± SE of maximal contraction amplitude (as a percentage of resting cell length); n, no. of cells.

^P < 0.025, KO-GFP vs. KO-PLM myocytes.

Table 5.

[Ca^]_i transients in PLM KO myocytes infected with advenovirus containing PLM and cultured for 48 h

	KO-GFP		KO-PLM
	Means ± SE	n	Means ± SE	n
Systolic [Ca]i, nM
1.8 mM [Ca]o	191±6	33	175±16	13
5.0 mM [Ca]o	301±93	54	224±12*	23
Diastolic [Ca]i, nM
1.8 mM [Ca]o	98±4		85±10
5.0 mM [Ca]o	95±4		86±7
[Ca]i transient amplitude, %increase in fluorescence
1.8 mM [Ca]o	18.2±0.6		18.6±1.0
5.0 mM [Ca]o	37.1±0.9		27.1±1.0*
t1/2 of [Ca]i decline, ms
1.8 mM [Ca]o	191±12		186±9
5.0 mM [Ca]o	149±5		163±9

Values are means ± SE; n, no. of cells. [Ca^]_i, cytosolic Ca ^{concentration;} t_1/2, half-time. The results of two-way ANOVA were as follows: for systolic [Ca^]_i, group (P < 0.0001), [Ca^]_o (P < 0.0001), and group × [Ca^]_o interaction effects (P < 0.009); for diastolic [Ca^]_i, group (P < 0.13), [Ca^]_o (P < 0.49), and group × [Ca^]_o interaction effects (P < 0.43); for [Ca^]_i transient amplitude, group (P < 0.0001), [Ca^]_o (P < 0.0001), and group × [Ca^]_o interaction effects (P < 0.0001); and for t_1/2 of [Ca^]_i decline, group (P < 0.55), [Ca^]_o (P < 0.002), and group × [Ca^]_o interaction effects (P < 0.33).

^P < 0.01, KO-GFP vs. KO-PLM myocytes.

Table 6.

Effects of PLMS68E and PLMS68A mutants on PLM KO myocytes

	KO-GFP		KO-PLM		KO-S68E		KO-S68A
	Means ± SE	n	Means ± SE	n	Means ± SE	n	Means ± SE	n
Maximal contraction amplitude, %resting cell length
1.8 mM [Ca]o	4.91±0.33	16	5.16±0.40	4	5.02±0.27	15	4.92±0.28	13
5.0 mM [Ca]o	8.99±0.22	26	7.35±0.33*	13	7.19±0.21*	28	8.73±0.41	19
[Ca]i transient amplitude, %increase in fluorescence
1.8 mM [Ca]o	18.2±0.6	33	18.6±1.0	13	17.9±1.2	16	17.8±1.3	17
5.0 mM [Ca]o	37.1±0.9	54	27.1±1.0*	23	26.4±1.3*	22	36.2±1.0	12
t1/2 of [Ca]i decline, ms
1.8 mM [Ca]o	191±12		186±9		188±10		195±17
5.0 mM [Ca]o	149±5		163±9		174±6		159±14
SR Ca content, fmol/fF
5.0 mM [Ca]o	12.1±0.2	4	5.4±1.6*	5	6.0±0.9*	8	11.3±0.9	6

Values are means ± SE; n, no. of cells. Contractility, [Ca^]_i transients, t_1/2 of [Ca^]_i decline, and sarcoplasmic reticulum (SR) Ca ^{contents were measured after adenovirus infection and 48 h of culture. For comparison, SR Ca content measured at 5 mM [Ca]}_o was 14.3 ± 0.6 fmol/fF (n = 9) in freshly isolated PLM KO myocytes (22). KO-S68E and KO-S68A, PLM KO myocytes infected with adenovirus expressing PLMS68E and PLMS68A mutants, respectively.

^P < 0.025 compared with KO-GFP myocytes.

Culture and adenoviral infection of adult mouse myocytes.

In our hands, adult mouse myocytes survived better in culture when plated on laminin- rather than Matrigel-coated coverslips. Cultured mouse myocytes maintained their elongated rod shapes when cultured in FBS for 48 h. With serum-free culture, the inclusion of BDM, ITS supplement, and BSA in MEM gave the best results in terms of the maintenance of rod shapes (∼65%) after 48 h (Fig. 1). When infected with adenovirus expressing GFP, green fluorescence started to appear by 18–24 h and increased throughout 48 h of culture (Fig. 1). With few exceptions, GFP fluorescence was much more intense in rod-shaped myocytes compared with rounded and hypercontracted myocytes. Other agents that inhibited myocyte contraction [blebbistatin (5.7 μM) or cytochalasin D (2 μM)] were not as effective as BDM in terms of maintaining myocyte viability in culture. The required presence of BDM precluded pacing adult mouse myocytes while in culture.

Fig. 1.

Culture and adenoviral infection of adult mouse myocytes. Cardiac myocytes were isolated from adult congenic phospholemman (PLM) knockout (KO) mice, infected with adenovirus expressing both green fluorescent protein (GFP) and the PLMS68A mutant, and placed in culture as described in methods. A: transmitted light image of adult mouse myocytes after 24 h of culture. B: fluorescent image of A demonstrating the expression of GFP. C: transmitted light image of another adult mouse myocyte culture at 48 h. D: fluorescent image of C. Both B and D are raw images and were taken at the same time using identical excitation light intensities, with the camera set at the same gain but with autoexposure mode. The higher background in B compared with D is due to the longer exposure time because of the much lower GFP fluorescence emission from adenovirus-infected myocytes cultured for 24 h. In contrast, GFP fluorescence emission from infected myocytes cultured for 48 h was much stronger and required a shorter exposure time, as indicated by the lower background.

Effects of culture and adenoviral infection on whole cell membrane capacitance, t-tubules, and selected proteins involved in excitation-contraction coupling.

Membrane capacitance (C_m) in PLM KO myocytes cultured for 48 h (180 ± 5 pF, n = 27) was not significantly (P < 0.065) different than that measured in freshly isolated PLM KO myocytes (164 ± 5 pF, n = 50), suggesting that myocyte surface membrane area did not appreciably change with short-term culture. When di-8-ANEPPS was used to stain surface sarcolemma and t-tubules, myocytes cultured for 24 and 48 h retained their t-tubular network compared with freshly isolated myocytes (Fig. 2). Pretreatment with formamide resulted in a complete loss of t-tubules (not shown).

Fig. 2.

Effects of culture on t-tubules in mouse cardiac myocytes. Cardiac myocytes were isolated from wild-type (WT) C57BL/6 mice, plated on laminin-coated coverslips, and cultured for 24–48 h. Myocytes were stained with di-8-ANEPPS and imaged with both conventional wide-field fluorescence (A–F) and confocal (G–I) microscopes. Images acquired with ×10 (A–C), ×60 (D–F), and ×100 (G–I) objectives of myocytes at day 0 (A, D, and G), day 1 (B, E, and H), and day 2 (C, F, and I) of culture are shown.

There were no significant differences in the protein levels of NCX1, the α₁-subunit of Na^{-K-ATPase, SERCA2, and calsequestrin between freshly isolated WT and PLM KO myocytes (}Fig. 3 and Table 1), in agreement with our previous results (22) and those of others (2). After 48 h of culture, PLM KO myocytes retained similar amounts of NCX1 and SERCA2 but suffered an ∼18% loss in the α₁-subunit of Na^{-K-ATPase compared with freshly isolated PLM KO myocytes (}Fig. 3 and Table 1). Infection of PLM KO myocytes with adenovirus expressing either GFP or WT PLM followed by culture for 48 h did not have any appreciable effects on the protein levels of NCX1 and SERCA2 but resulted in a similar reduction in the α₁-subunit of Na^{-K-ATPase compared with freshly isolated myocytes (}Fig. 3 and Table 1).

Fig. 3.

Effects of culture on selected proteins involved in excitation-contraction coupling. Myocyte homogenates were prepared from day 0 WT (WT-D0), day 0 PLM KO (KO-D0), day 2 PLM KO (KO-D2), day 2 GFP-expressing KO (KO-GFP), and day 2 PLM-expressing KO (KO-PLM) myocytes and subjected to SDS-PAGE followed by Western blot analysis. Primary antibodies (methods) were used to detect cardiac Na^{/Ca exchanger (NCX1), the α}₁-subunit of Na^{-K-ATPase, sarco(endo)plasmic reticulum Ca-ATPase 2 (SERCA2), and calsequestrin (CLSQ). Composite results are shown in} Table 1.

PLM KO myocytes, when infected with adenovirus expressing either WT PLM or its Ser ^{mutants, expressed the constructs robustly after 48 h of culture (}Fig. 4). WT PLM, either endogenous in WT-GFP myocytes or exogenous in KO-PLM myocytes, was partially phosphorylated, as detected by the phospho-specific CP68 antibody (Fig. 4). In addition, protein levels of both phosphorylated and unphosphorylated exogenous PLM in KO-PLM myocytes were much higher than those present in WT-GFP myocytes.

Fig. 4.

Expression of WT PLM and its Ser ^{mutants in cultured PLM KO myocytes. Adult cardiac myocytes isolated from congenic PLM KO mice were infected with adenovirus expressing either GFP alone (KO-GFP), GFP + WT dog PLM (KO-PLM), GFP + PLMS68A (KO-S68A), or GFP + PLMS68E (KO-S68E) and cultured for 48 h. Myocytes from WT littermates infected with adenovirus expressing GFP (WT-GFP) and cultured for 48 h were used as controls. Proteins in myocyte lysates (50 μg/lane) were separated by gel electrophoresis and transferred to ImmunBlot polyvinylidene difluoride membranes. Endogenous mouse and WT dog PLM, unphosphorylated and phosphorylated at Ser, was probed with C2 and CP68 anti-PLM antibodies, respectively, whereas dog PLM Ser mutants were detected with B8 anti-PLM antibody. C2 antibody recognizes predominantly the unphosphorylated COOH-terminus of both dog and rodent PLM, and CP68 antibody is specific for PLM phosphorylated at Ser (}16, 26), whereas B8 antibody recognizes the NH₂-terminus of dog but not rodent PLM (18).

Effects of culture on adult mouse myocyte contractility.

After the removal of BDM, cultured adult mouse myocytes contracted at the externally imposed pacing frequency. In contrast to freshly isolated myocytes, most mouse myocytes cultured for 48 h failed to contract when stimulated at 0.6 mM [Ca^]_o. This phenomenon made it very difficult to precisely quantify contraction amplitudes at 0.6 mM [Ca^]_o. For this reason, in this study, we only examined contraction and [Ca^]_i transients at 1.8 and 5.0 mM [Ca^]_o.

As shown in Table 2, WT and PLM KO myocytes cultured for 24 and 48 h in serum-free media maintained contraction amplitudes that were slightly less than those observed for freshly isolated myocytes (day effect, P < 0.035). As expected, elevating [Ca^]_o increased contraction amplitudes ([Ca^]_o effect, P < 0.0001). In addition, contraction amplitudes were different between cultured WT and PLM KO myocytes (group effect, P < 0.003). Specifically, contraction amplitudes in PLM KO myocytes were significantly higher than WT myocytes at 5.0 but not 1.8 mM [Ca^]_o (group × [Ca^]_o interaction effect, P < 0.03). Since freshly isolated myocytes from PLM KO hearts exhibited contraction amplitudes that were similar at 1.8 mM [Ca^]_o but higher at 5.0 mM [Ca^]_o compared with WT myocytes (22), our present observation suggests that the contractile differences between WT and PLM KO myocytes were preserved after 48 h of culture.

Effect of adenoviral infection on cultured myocyte contractility.

Infection with recombinant, replication-deficient adenovirus has been shown to be highly efficient for gene delivery to adult cardiac myocytes (17, 21, 29). Indeed, >95% of rod-shaped adult mouse myocytes exhibited robust GFP fluorescence after an infection with Adv-GFP (Fig. 1). Compared with uninfected myocytes, PLM KO myocytes infected with Adv-GFP had similar cell shortening amplitudes after 24 and 48 h of culture, regardless of whether contraction was measured with 1.8 or 5.0 mM [Ca^]_o (Table 3). This conclusion was supported by three-way ANOVA, which indicated insignificant group (PLM KO vs. KO-GFP, P < 0.80) and day (P < 0.11) effects but significant [Ca^]_o (P < 0.0001) effects. There were no significant interaction effects.

Rescue of PLM deficiency in PLM KO myocytes.

Since PLM KO myocytes maintained their contractile phenotype after short-term culture (Table 2), we next determined if the contractile phenotype would revert back to that observed for WT myocytes after “rescue” with PLM. Compared with KO-GFP myocytes, contraction amplitudes in KO-PLM myocytes were similar at 1.8 mM [Ca^]_o but lower at 5.0 mM [Ca^]_o (Fig. 5 and Table 4; group × [Ca^]_o interaction effect, P < 0.025). There were no differences in contraction amplitudes between WT and KO-PLM myocytes, indicating that PLM rescue was successful in restoring the contractile phenotype in PLM KO myocytes back to that observed in WT myocytes. [Ca^]_i transients in PLM KO myocytes were similarly affected by PLM overexpression: systolic [Ca^]_i and [Ca^]_i transient amplitudes were similar at 1.8 mM [Ca^]_o but significantly lower at 5.0 mM [Ca^]_o (Fig. 5 and Table 5). The half-life (t_1/2) of [Ca^]_i decline, an estimate of in situ SR Ca ^{uptake activity (}27), was not different between KO-PLM and KO-GFP myocytes (Table 5), indicating that PLM had no effect on SERCA2 activity. In support of this interpretation, there were no differences in SERCA2 protein levels between KO-GFP and KO-PLM myocytes (Fig. 3 and Table 1). Importantly, both I_NaCa (Fig. 6) and I_pump (Fig. 7) were significantly suppressed in KO-PLM myocytes compared with KO-GFP myocytes. Compared with WT myocytes, both I_NaCa (Fig. 6) and I_pump (Fig. 7) were higher in KO-GFP but much lower in KO-PLM myocytes. Severe suppression of I_NaCa and I_pump in KO-PLM myocytes is likely due to the supraphysiological levels of both phosphorylated and unphosphorylated exogenous PLM in KO-PLM myocytes compared with the endogenous levels of PLM found in WT myocytes (Fig. 4). Despite no differences in SR Ca ^{uptake activity and SERCA2 protein levels, SR Ca content measured at 5 mM [Ca]}_o was higher in KO-GFP compared with KO-PLM myocytes (Table 6).

Fig. 5.

Effects of PLM and its Ser ^{mutants on contractility and cytosolic Ca concentration ([Ca]}_i) transients in cultured PLM KO myocytes. Adult cardiac myocytes isolated from congenic PLM KO myocytes were infected with adenovirus expressing either GFP alone (A and E), GFP + WT dog PLM (B and F), GFP + PLMS68A (C and G), or GFP + PLMS68E (D and H) and cultured for 48 h. Myocytes were incubated at 5 mM extracellular Ca ^{concentration at 37°C and paced to contract at 1 Hz. Steady-state myocyte contractions (}A–D) and [Ca^]_i transients (E–H) are shown. Composite data are shown in Tables 4–6.

Fig. 6.

Effects of WT PLM and its Ser ^{mutants on Na/Ca exchange current (}I_NaCa) in cultured PLM KO myocytes. Adult cardiac myocytes from congenic PLM KO mice were infected with adenovirus expressing either GFP alone, GFP + WT PLM, GFP + PLMS68A, or GFP + PLMS68E. After myocytes had been cultured for 48 h, I_NaCa was measured as described in methods. Current density-voltage relationships of I_NaCa (means ± SE) from KO-GFP (□; n = 11), KO-PLM (▴; n = 11), KO-S68A (•; n = 7), and KO-S68E (○; n = 9) myocytes are shown. For comparison, current density-voltage relationships of I_NaCa from WT (⧫; n = 20) myocytes are also shown. The reversal potential of I_NaCa in all myocytes examined was approximately −60 mV. Note that I_NaCa was lower in WT compared with KO-GFP myocytes. In addition, I_NaCa was suppressed by WT PLM and the PLMS68E mutant but not by the PLMS68A mutant. Error bars are not shown if they fall within the boundaries of the symbol.

Fig. 7.

Effects of WT PLM and its Ser ^{mutants on Na-K-ATPase current (}I_pump) in cultured PLM KO myocytes. Adult myocytes from congenic PLM KO hearts were infected with adenovirus expressing either GFP alone, GFP + WT PLM (PLM), GFP + PLMS68A (S68A), or GFP + PLMS68E (S68E). After 48 h of culture, I_pump was measured at 18 mM extracellular K ^{concentration at 0 mV at 30°C with pipette Na concentrations ([Na]}_pip) of either 10 mM (open bars) or 80 mM (solid bars). For comparison, I_pump of WT myocytes is shown. At 10 mM [Na^]_pip, there were 8 WT, 9 KO-GFP, 10 KO-PLM, 6 KO-S68A, and 7 KO-S68E myocytes. At 80 mM [Na^]_pip, there were 5 WT, 7 KO-GFP, 6 KO-PLM, 8 KO-S68A, and 8 KO-S68E myocytes. Two-way ANOVA indicated significant differences between WT and KO-GFP (P ≤ 0.05), WT and KO-PLM (P < 0.0004), WT and KO-S68A (P < 0.001), KO-GFP and KO-PLM (P < 0.002), KO-GFP and KO-S68A (P < 0.003), KO-PLM and KO-S68E (P < 0.003), and KO-S68A and KO-S68E (P < 0.004) myocytes, but not between KO-GFP and KO-S68E (P < 0.8), KO-PLM and KO-S68A (P < 0.8), and WT and KO-S68E (P < 0.3) myocytes.

Effects of the PLMS68E mutant on contractility, [Ca^]_i transients, SR Ca ^{uptake, SR Ca content, I}_NaCa, and I_pump on PLM KO myocytes.

Previous studies have indicated that PLM, when phosphorylated at Ser^{, strongly inhibits NCX1 (}18, 25) while it simultaneously relieves its inhibition on Na^{-K-ATPase (}8, 10, 11, 16). To investigate whether the regulation of myocyte contractility by PLM is mediated via its effects on NCX1 versus Na^{-K-ATPase, we used the phosphomimetic PLM mutant S68E, which has been shown to inhibit} I_NaCa in adult rat cardiac myocytes (18) and transfected HEK-293 cells (25). In PLM KO myocytes devoid of endogenous PLM, overexpression of the PLMS68E mutant resulted in a large suppression of I_NaCa (Fig. 6). Because phosphorylated PLM has been reported to either lower the K_m for Na ⁽8) or increase the V_max of Na^{-K-ATPase (}16, 26), we measured I_pump at [Na^]_pip of 10 mM (K_m effect) and 80 mM (V_max effect). Overexpression of the PLMS68E mutant in PLM KO myocytes did not affect I_pump (Fig. 7; group effect, P < 0.35, and group × [Na^]_pip interaction effect, P < 0.80). At 5.0 mM [Ca^]_o, contraction amplitudes of KO-S68E myocytes were significantly (P < 0.0004) lower than those of KO-GFP myocytes but similar (P < 0.99) to those of KO-PLM myocytes (Fig. 5 and Table 6). Similarly, [Ca^]_i transient amplitudes of KO-S68E myocytes measured at 5.0 mM [Ca^]_o were lower compared with those of KO-GFP myocytes (Fig. 5 and Table 6). There were no differences in SR Ca ^{uptake, although SR Ca content was significantly lower in KO-S68E compared with KO-GFP myocytes (}Table 6).

Effects of the PLMS68A mutant on contractility, [Ca^]_i transients, SR Ca ^{uptake, SR Ca content, I}_NaCa, and I_pump on PLM KO myocytes.

The PLMS68A mutant has been shown to have no effect on I_NaCa in adult rat cardiac myocytes (18) and in transfected HEK-293 cells (25). Overexpression of the PLMS68A mutant in PLM KO myocytes had no effect on I_NaCa (Fig. 6) but resulted in significant inhibition of I_pump (Fig. 7), which appeared to be more prominent at [Na^]_pip of 80 mM (∼66% inhibition) than 10 mM (∼51% inhibition). This conclusion was supported by two-way ANOVA, which indicated significant group (P < 0.0001), [Na^]_pip (P < 0.0001), and group × [Na^]_pip interaction (P < 0.0025) effects. Our results suggest that unphosphorylated PLM regulates Na^{-K-ATPase primarily by a} V_max effect, although a K_m effect cannot be excluded. Contraction and [Ca^]_i transient amplitudes in KO-S68A myocytes were not different than those observed in KO-GFP myocytes, whether measured at 1.8 or 5.0 mM [Ca^]_o (Fig. 5 and Table 6). Similarly, there were no differences in SR Ca ^{uptake and SR Ca content (}Table 6) between KO-GFP and KO-S68A myocytes.

DISCUSSION

Current research on cardiac pathophysiology frequently makes use of genetically manipulated mouse models. Compared with adult rat hearts, it is technically more challenging to isolate viable myocytes from adult mouse hearts and maintain their viability in culture. In 2000, Zhou et al. (30) made a major contribution in establishing the experimental protocols for isolation, culture, and adenoviral infection of adult mouse cardiac myocytes. In 2003, O'Connell et al. (15) published a modified method for the isolation and culture of adult mouse cardiac myocytes. Although both studies demonstrated success in maintaining rod-shaped morphology in culture for up to 72 h (15, 30), functional studies (contraction, [Ca^]_i transients, ion currents, etc.) were only shown for myocytes cultured for 18–24 h. Our present study has extended these observations by demonstrating that myocyte contractility and [Ca^]_i homeostasis were well preserved at 48 h. In addition, myocyte surface membrane area, t-tubule organization, and protein levels of NCX1, SERCA2, and calsequestrin were not affected after 48 h of culture. One caveat is that unlike freshly isolated myocytes (22), the majority of adult mouse myocytes cultured for 48 h failed to contract when [Ca^]_o was lowered to 0.6 mM. Another change brought on by short-term culture is the small reduction (∼18%) in the α₁-subunit of Na^{-K-ATPase. [Ca]}_i transients and ion currents, however, were easily measurable in adult mouse cardiac myocytes after 48 h of culture. Another important finding is that adenovirus-mediated gene transfer in adult mouse cardiac myocytes resulted in the robust expression of the exogenous gene with functional consequences after 48 h of culture. In our hands, the major factors for a successful outcome are gentle and careful isolation procedures to ensure a maximum yield of healthy rod-shaped myocytes, lowering of pH in culture from 7.40 to 7.00, inclusion of ITS supplement and BSA in the culture medium, and the absolute requirement of BDM during myocyte isolation and culture. We have tried other inhibitors of excitation-contraction coupling, such as blebbistatin and cytochalasin D, without much success.

Overexpression (17) and downregulation (14) or elimination (22) of PLM have profound effects on cardiac myocyte contractility and [Ca^]_i transients. Since PLM regulates both Na^{-K-ATPase (}8, 10, 16, 26) and NCX1 (22, 25, 28) in the heart, it is difficult to dissect which ion transport pathway is the primary mediator of PLM's effect on myocyte contractility. To approach the issue of whether inhibition of Na^{-K-ATPase or NCX1 represents the major mechanism by which PLM regulates cardiac contractility, we used myocytes isolated from PLM KO hearts in which there was no endogenous PLM. A major finding of our present study is that contraction differences between PLM KO and WT mouse myocytes were preserved after 48 h of culture. Another important finding is that adenovirus infection, by itself, did not affect myocyte contractility examined after 24 and 48 h of culture. These two observations made it possible to express PLM or its mutants in PLM KO myocytes to examine the effects on contractility, [Ca]}_i transients, I_pump, and I_NaCa. The reintroduction of WT PLM into cultured PLM KO myocytes had no effects on the protein levels of NCX1, the α₁-subunit of Na^{-K-ATPase, SERCA2, and calsequestrin compared with matched PLM KO myocytes overexpressing GFP. Contraction and [Ca]}_i transient amplitudes in KO-PLM myocytes, however, were restored to levels observed in WT myocytes. In addition, both I_pump and I_NaCa in PLM KO myocytes were suppressed by WT PLM overexpression. These results indicate that PLM has an important role in the regulation of cardiac contractility and [Ca^]_i homeostasis in resting myocytes, and the mechanism may involve modulating Na^{-K-ATPase and/or NCX1 activities. Despite no change in SERCA2 levels and SR Ca uptake activity, SR Ca content was lower in KO-PLM compared with KO-GFP myocytes. This observation suggests that altered SR Ca content is likely the result of inhibition of Na/Ca exchange by PLM, either directly or indirectly through its action on Na-K-ATPase. The lower SR Ca content in KO-PLM myocytes largely accounts for the decreased [Ca]}_i transient and contraction amplitudes.

To differentiate the effects of PLM on Na^{-K-ATPase and/or NCX1, we used the PLMS68E mutant, which inhibited} I_NaCa but not I_pump when overexpressed in PLM KO myocytes. PLMS68E overexpression resulted in a depression of contraction and [Ca^]_i transient amplitudes at 5.0 but not 1.8 mM [Ca^]_o compared with matched PLM KO myocytes expressing GFP. This phenotype is characteristic of myocytes in which NCX1 was downregulated (21). In addition, SR Ca ^{content was lower in KO-S68E myocytes despite no changes in SR Ca uptake. These observations suggest that direct inhibition of NCX1 by phosphorylated PLM resulted in decreased SR Ca content, leading to reduced [Ca]}_i transient and contraction amplitudes. Conversely, when we overexpressed the PLMS68A mutant, which inhibited I_pump but not I_NaCa in PLM KO myocytes, there were no differences in SR Ca ^{uptake, SR Ca content, contraction, and [Ca]}_i transient amplitudes compared with matched PLM KO myocytes infected with Adv-GFP. Our results clearly indicate that in isolated myocytes studied under our conditions, the effects of PLM on cardiac contractility were mediated largely by its inhibitory effects on NCX1 rather than Na^-K-ATPase.

Some insight into the mechanism by which PLM regulates cardiac contractility can also be gleaned from the distribution of the isoforms of Na^{-K-ATPase, NCX1, and PLM in adult heart muscle. Whereas PLM and NCX1 are distributed in both sarcolemma and t-tubules (}28), the α₁-isoform (ouabain insensitive) and α₂-isoform (ouabain sensitive) of Na^{-K-ATPase are preferentially distributed in the sarcolemma and t-tubules, respectively (}3, 20). In rodent hearts, PLM associates with and modulates the activity of the α₁-isoform but not α₂-isoform of Na^{-K-ATPase (}16), although both α₁- and α₂-isoforms of Na^{-K-ATPase coimmunoprecipitate with PLM in bovine (}6) and rabbit (4) cardiac tissues. In adult rat cardiac myocytes, inhibition of the α₂-isoform of Na^{-K-ATPase results in increased} I_NaCa and enhanced cardiac myocyte contractility (20), indicating intimate cross-talk between the α₂-isoform of Na^{-K-ATPase and NCX1 in the t-tubules. Similar intimate cross-talk between NCX1 and the α}₂-isoform of Na^{-K-ATPase has also recently been demonstrated for mouse aortic smooth muscle cells (}13). The observations that in rodent cardiac myocytes PLM regulates the α₁-isoform but not α₂-isoform of Na^{-K-ATPase, and that cross-talk occurs predominantly between the α}₂-isoform of Na^{-K-ATPase and NCX1 in the t-tubules, support our hypothesis that PLM regulates cardiac contractility by mechanisms other than inhibition of Na-K-ATPase. Our results with the PLMS68A mutant expressed in PLM KO myocytes, showing that it inhibited} I_pump but had no effects on contraction and [Ca^]_i transient amplitudes, can be explained by the concept that in rodent cardiac myocytes, PLM inhibits the α₁-isoform but not α₂-isoform of Na^{-K-ATPase (}16). Definitive proof of this hypothesis, however, will require further investigation with mouse cardiac myocytes.

In PLM KO myocytes cultured for 48 h and expressing GFP, reducing [Na^]_pip from saturating 80 to 10 mM resulted in an ∼66% decrease in I_pump, suggesting tht the K_m for Na ^{in PLM KO myocytes was ∼10 mM. This interpretation is consistent with the findings that the} K_m for Na ^{was 14.6 ± 1.9 mM in freshly isolated PLM KO myocytes (}8) and ∼10 mM in freshly isolated adult rat myocytes (26). When the PLMS68A mutant was expressed in PLM-KO myocytes to simulate maximal inhibition of Na^{-K-ATPase by unphosphorylated WT PLM, the major effect was a decrease in the maximal amplitude of} I_pump. This V_max effect of PLM on I_pump is in agreement with the observations in adult rat myocytes overexpressing PLM (26) and in freshly isolated guinea pig myocytes stimulated with forskolin (16). A comparison of Na^{-K-ATPase activities measured under saturating Na conditions in heart homogenates obtained from WT and PLM KO mice also indicated a} V_max effect of PLM (2). In contrast, when pump activity was measured as the Na^{-K-ATPase-mediated [Na]}_i decline in Na^{-loaded mouse myocytes, phosphorylating Ser of PLM resulted in a decrease in the} K_m for Na ^{with little effect on the} V_max of Na^{-K-ATPase (}8). Only when both Ser ^{and Ser of PLM were phosphorylated was an increase in} V_max evident in mouse myocytes (11). These results, when viewed together, suggest that PLM affects both the K_m for Na ^and V_max of Na^{-K-ATPase in rodent cardiac myocytes.}

There are limitations to the present study. The first limitation is that our measurements were exclusively performed in single cultured myocytes. Therefore, caution must be exercised when extrapolating our findings to the intact heart. A good example of this is that despite evidence of hypertrophy in PLM KO hearts (2, 12), neither we (22) nor Despa et al. (8) could detect hypertrophy in myocytes isolated from PLM KO hearts. Indeed, whether contractile performance in PLM KO hearts was superior (12) or depressed (2) in the intact animal remains controversial at the present time. The second limitation is that contractility of cultured adult mouse myocytes, although preserved, was not quite “normal,” as evidenced by the high incidence of contractile failure at 0.6 mM [Ca^]_o, and contraction amplitudes did decline slightly with increasing days in culture. In addition, protein levels of the α₁-subunit of Na^{-K-ATPase were slightly depressed compared with freshly isolated myocytes. The third limitation is that the reintroduction of WT PLM into PLM KO myocytes by adenovirus-mediated gene transfer resulted in higher than physiological levels of PLM. The supraphysiological levels of both phosphorylated and unphosphorylated PLM in infected myocytes, compared with PLM levels naturally present in WT controls, resulted in a severe suppression of} I_NaCa and I_pump in KO-PLM myocytes compared with WT myocytes. Finally, the interpretation of our results relies on the assumption that PLMS68A and PLMS68E mutants faithfully mimic the unphosphorylated and phosphorylated forms of PLM, respectively. This appears reasonable since in HEK-293 cells coexpressing NCX1 and WT PLM or its mutants, PLMS68E but not PLMS68A mimicked the inhibitory effects of forskolin on I_NaCa (25).

In summary, adult mouse myocytes were successfully maintained in culture for 48 h with near-normal contractility; no changes in membrane surface area, t-tubular organization, SERCA2, NCX1, and calsequestrin levels; and a slight (∼18%) decrease in α₁-subunit of Na^{-K-ATPase levels. Infection with adenovirus did not affect myocyte contractility. Contractility differences between WT and PLM KO myocytes were preserved after 48 h in culture. Overexpression of WT PLM in PLM KO myocytes had no effect on NCX1 and SERCA2 levels, and SR Ca uptake, but} I_NaCa, I_pump, and SR Ca ^{content were decreased, and resulted in contraction amplitudes indistinguishable from those of cultured WT myocytes. Overexpression of the phosphomimetic mutant of PLM (S68E) in PLM KO myocytes inhibited} I_NaCa but not I_pump or SR Ca ^{uptake and resulted in decreased SR Ca content, contraction, and [Ca]}_i transient amplitudes compared with PLM KO myocytes. In contrast, overexpression of the PLMS68A mutant (which cannot be phosphorylated) in PLM KO myocytes inhibited I_pump but not I_NaCa or SR Ca ^{uptake and had no effects on SR Ca content, contraction, and [Ca]}_i transient amplitudes compared with PLM KO myocytes. We conclude that in single cardiac myocytes, the regulation of contractility and [Ca^]_i transients by PLM was mediated by its effects on NCX1 rather than Na^-K-ATPase.

GRANTS

This work was supported in part by National Institutes of Health Grants RO1-HL-58672 and RO1-HL-74854 (to J. Y. Cheung), RO1-DK-46678 (to J. Y. Cheung, coinvestigator), RO1-HL-69074 (to A. L. Tucker), and PO1-HL-91799 (to A. M. Feldman) and by an American Heart Association Scientist Development Grant (to T. O. Chan).