Induced overexpression of phospholemman S68E mutant improves cardiac contractility and mortality after ischemia-reperfusion

Abstract

Phospholemman (PLM), when phosphorylated at Ser(68), inhibits cardiac Na+ / Ca2+ exchanger 1 (NCX1) and relieves its inhibition on Na+ -K+ -ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8-10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ∼35-75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+ -ATPase, α1- and α2-subunits of Na+ -K+ -ATPase, α1c-subunit of L-type Ca2+ channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ∼47% but the NCX1 current was depressed by ∼34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+ -K+ -ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+ concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+ contents, and [Ca(2+)]i transient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+ content, and [Ca2+]i transient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/dt in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/dt and -dP/dt and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.

Keywords: FXYD proteins; in vivo hemodynamics; intracellular Ca2+ regulation; ischemic cardiomyopathy.

Fig. 1.

Immunoblots of phospholemman (PLM) and S68E mutant in hearts. A: left ventricular (LV) homogenates were prepared from dogs, wild-type (WT; tTA^+/−S68E^−/−) mice, and transgenic (tTA^+/−S68E^+/−) mice (8 wk old) induced to overexpress the dog PLM S68E mutant [induced (Ind) S68E] at 5 wk of age and subjected to SDS-PAGE followed by Western blot analysis. B8 antibody (1:2,000), which recognizes the NH₂-terminus of dog PLM (34) but not rat or mouse (33, 42) PLM, was used to detect dog PLM and the S68E mutant in WT and induced hearts (50 μg/lane). C2 antibody (1:10,000), which was raised against the COOH-terminus of rat PLM (35), was used to detect signals from dog, WT, and induced hearts (3 μg/lane). Calsequestrin (CLSQ) was used as the loading control. Heart homogenates were subjected to alkaline phosphatase treatment to dephosphorylate PLM before being probed with C2 antibody (46). B: phospho-specific PLM antibody CP68 (1:2,000) (29) was used to detect PLM phosphorylated at Ser⁶⁸ in heart homogenates prepared from mice constitutively overexpressing the S68E mutant (ConS68E; 2 μg/lane), WT (20 μg/lane) mice, and noninduced (NonInd; 20 μg/lane) mice. In constitutively overexpressing mice, endogenous PLM represents <2.5% of the C2 signal (33). The absence of the CP68 signal in constitutively overexpressed samples indicates that CP68 does not detect the S68E mutant. As a control, C2 antibody (1:10,000) was used to detect unphosphorylated PLM or the S68E mutant in alkaline phosphatase-pretreated constitutively overexpressing (0.5 μg/lane), WT (5 μg/lane), and noninduced heart homogenates (5 μg/lane). C: myocytes isolated from induced and noninduced mice were exposed to vehicle or isoproterenol (Iso; 1 μM) for 6 min, after which they were processed for Western blot analysis. In this experiment, myocyte lysates were not treated with alkaline phosphatase before being probed with C2 (5 μg/lane, 1:10,000) or CP68 (20 μg/lane, 1:2,000) antibodies.

Fig. 2.

Induced overexpression of the S68E mutant does not result in mortality. Survival curves of WT mice (n = 20), mice constitutively overexpressing the S68E mutant (n = 15), mice induced at 5 wk to express the S68E mutant (n = 10), and transgenic S68E mice kept on a doxycycline diet to prevent induction of the S68E mutant (noninduced; n = 15) are shown.

Fig. 3.

Induced expression of the S68E mutant does not result in arrhythmias. Echocardiography was performed in 8-wk-old noninduced mice (top) and mice induced to express the S68E mutant (middle). For comparison, echocardiography of 4-wk-old mice constitutively overexpressing the S68E transgene (bottom) is also shown. ECG tracings demonstrated normal sinus rhythm in both induced and noninduced mice but severe bradycardia and multifocal ventricular tachycardia in the mouse constitutively overexpressing the S68E mutant.

Fig. 4.

Induced expression of the S68E transgene enhances the in vivo contractility response to Iso. In vivo catheterization was performed in anesthetized mice (see methods), and the maximal first time derivatives of the left venticular (LV) pressure rise (+dP/dt) and fall (−dP/dt) and heart rate were continuously monitored, both at baseline and with increasing doses of Iso. The average maximal +dP/dt achieved with each dose of Iso in six noninduced mice (●) and eight induced mice (◇) is shown. Composite results are shown in Table 1. Two-way ANOVA indicated significant group (P < 0.0065) and Iso (P < 0.0001) effects. *P < 0.0065, noninduced vs. induced mice.

Fig. 5.

Induced expression of the S68E transgene enhances LV expression of the Na⁺/Ca²⁺ exchanger (NCX). LV homogenates were prepared from 8-wk-old WT, noninduced, and induced mice and subjected to SDS-PAGE followed by Western blot analysis (see methods). Protein loading was 50 μg/lane for all proteins. Primary antibodies were used to detect the cardiac ryanodine receptor phosphorylated at Ser²⁸⁰⁸ (p-RyR; 1:1,000), α_1c-subunit of the L-type Ca²⁺ channel (Ca_v1.2; 1:200), NCX1 (1:1,000), sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2; 1:5,000), α₁- (1:1,000) and α₂- (1:2,000) subunits of Na⁺-K⁺-ATPase, and CLSQ (1:3,000). Signal band intensities of each protein were normalized against the corresponding CLSQ loading control signal and are shown as composite data in Table 2.

Fig. 6.

Induced expression of the S68E mutant inhibits NCX current (I_NaCa). A: 3 wk after the induction of S68E expression by removing doxycycline in the diet, myocytes were isolated from the LV, and I_NaCa (Na⁺ concentration in the pipette: 12.25 mM, Ca²⁺ concentration in the pipette: 205 nM, extracellular Na⁺ concentration: 141.2 mM, and Ca²⁺ concentration: 5 mM) was measured at 30°C (, , –42, 45). The reversal potential of I_NaCa in all myocytes examined was −60 mV, close to the theoretical reversal potential of −73 mV under prevailing ionic conditions. I_NaCa was significantly (P < 0.0001, group × voltage interaction effects) lower in myocytes induced to express the S68E mutant (open squares; n = 11) compared with noninduced myocytes (open circles; n = 13). Error bars are not shown if they fell within the boundaries of the symbol. B: in another series of experiments, I_NaCa was measured in induced (squares; n = 5) and noninduced (circles; n = 4) myocytes in the absence (open symbols) and presence (filled symbols) of 1 μM Iso. For clarity of presentation, lines are only drawn through open symbols.

Fig. 7.

Induced S68E expression had no effects on currents due to α₁-subunts (I_α1) and α₂-subunits (I_α2) of Na⁺-K⁺-ATPase. Currents due to α₁- and α₂-subunits of Na⁺-K⁺-ATPase (80 mM Na⁺ concentration in the pipette and 18 mM extracellular K⁺ concentration, 30°C) were separated by their differential sensitivities to dihydroouabain (43). Top and bottom: I_α1 and I_α2, respectively, in induced (n = 8) and noninduced (n = 8) myocytes both at baseline (open bars) and after stimulation with 1 μM Iso (solid bars).

Fig. 8.

Intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and cell shortening in induced and noninduced myocytes. A and B: representative traces of [Ca²⁺]_i transients (A) and contraction (B) in noninduced (left) and induced (right) myocytes paced at 2 Hz, 1.8 mM extracellular Ca²⁺ concentration ([Ca²⁺]_o), and 37°C. Iso, where indicated, was at 1 μM. Composite results are shown in Table 3.

Fig. 9.

Time course of contraction and systolic [Ca²⁺]_i changes in response to Iso. Top: myocyte contraction amplitudes (% of resting cell length) from induced (●; n = 10) and noninduced (○; n = 9) myocytes paced at 2 Hz, 1.8 mM [Ca²⁺]_o, and 37°C are shown. Iso (1 μM) was added at the times indicated. Bottom: systolic [Ca²⁺]_i from fura-2-loaded noninduced (○; n = 10) and induced (●; n = 11) myocytes paced at 2 Hz, 1.8 mM [Ca²⁺]_o, and 37°C are shown. After 2 min of pacing, steady-state [Ca²⁺]_i values were obtained (time 0) followed by the addition of Iso (1 μM), and measurements continued for 6.5 min.

Fig. 10.

Induced S68E expression increases sarcoplasmic reticulum (SR)-releasable Ca²⁺ content in response to Iso. Noninduced (A and C) and induced S68E (B and D) myocytes were incubated at 1.8 mM [Ca²⁺]_o and 30°C and voltage clamped at −90 mV. To ensure steady-state SR Ca²⁺ load, 12 conditioning pulses (from −90 to 0 mV, 300 ms, 1 Hz) were delivered before caffeine (5 mM, 200 ms after the 12th conditioning pulse) was puffed on the myocyte for 2.4 s, both in the absence (A and B) and presence (C and D) of Iso (1 μM). A large transient inward current caused by caffeine-induced SR Ca²⁺ release was observed. This current represents Na⁺ entry accompanying Ca²⁺ extrusion by NCX1, and the half-time (t_1/2) of current decline is a functional readout of Na⁺/Ca²⁺ exchange activity. In addition, the time integral of this current provides an estimate of SR-releasable Ca²⁺ (36, 37). 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 1 Ca²⁺. SR Ca²⁺ content was normalized to cell size (in fmol/fF). Results are shown in Table 3.

Fig. 11.

Infarct sizes were similar in noninduced and induced S68E hearts subjected to ischemia/reperfusion (I/R). Hearts were subjected to 30 min of ischemia followed by reperfusion for 3 days (see methods). Top: area at risk (AAR) [2,3,5-triphenyltetrazolium (TTC) positive and TTC negative], infarct area (TTC negative), and area not at risk (Evans blue dye stained) were determined for both noninduced and induced hearts. Bottom: summary for AAR and infarct size for noninduced I/R (n = 6) and induced I/R (n = 7) hearts. There were no differences in AAR (P < 0.90) and infarct size (P < 0.79) between noninduced and induced hearts.

Fig. 12.

Cardiac performance after I/R was better in induced compared with noninduced hearts. Hearts were subjected to sham operation (sham) or 30 min of ischemia followed by 3 days of reperfusion. In vivo catheterization was performed in anesthetized mice. Both +dP/dt and −dP/dt were continuously measured, both at baseline and at increasing doses of Iso. There were 7 WT sham mice (□), 7 noninduced I/R mice (●), and 11 induced I/R mice (◇), respectively. Error bars are not shown if they fell within the boundaries of the symbol. Composite results are shown in Table 1. Two-way ANOVA indicated P < 0.0003 for WT sham vs. noninduced I/R mice and P < 0.003 for induced I/R vs. noninduced I/R mice. There were no differences (P < 0.84) in +dP/dt between WT sham and induced I/R mice. *P < 0.003, noninduced I/R vs. induced I/R or WT sham mice.