Cardiac hypertrophy in mice expressing unphosphorylatable phospholemman

Abstract

Aims: Elevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force-frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM(3SA) knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression.

Methods and results: In wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM(3SA) mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na(+)]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM(3SA) myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM(3SA) mice compared with banded WT.

Conclusions: Decreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure.

Keywords: Hypertrophy; Intracellular sodium; Na/K ATPase; Phospholemman.

Figure 1

Protein expression and phosphorylation changes measured 8 weeks after aortic banding in C57BL/6J mice. Representative western blots (A) and quantitative changes in PLM/NKA α1 ratio and PLM phosphorylation (B). PLM phosphorylation was normalized to total PLM. (C) Changes in the expression of NCX and SERCA2a in sham and banded hearts. PLM, phospholemman; NKA α1, Na/K ATPase α1 subunit; CSQ, calsequestrin; NCX, sodium–calcium exchanger; SERCA2a, sarco/endoplasmic reticulum Ca ATPase type 2a. In A, the top and bottom panels show images of the same samples cropped from different gels. All other panels were each run on a single gel and images cropped within a gel (CSQ or total PLM was used as loading controls). Data in B and C are mean ± SEM, *P < 0.05; n = 6–11 (sham) and n = 6–10 (banded).

Figure 2

Protein expression and phosphorylation changes measured 3 and 14 days after aortic banding in C57BL/6J mice. Representative western blots (A) and quantitative changes (B) in PLM phosphorylation (Ser63 and 68); NCX and SERCA2a expression in sham and banded hearts, respectively. In A, each row represents images cropped from different gels probed with different antibodies as shown. Data are mean ± SEM, *P < 0.05; n = 6 (sham and banded).

Figure 3

Na/K pump current measurements in myocytes and cardiac function in Langendorf hearts isolated from sham and banded C57BL/6J mice. (A) Cell capacitance showing significant myocyte hypertrophy in the banded group, (B) original traces showing example Na/K pump current recordings (the downward deflections represent periods of K-free superfusion), (C) mean pump current density in cells isolated from sham and banded hearts normalized to cell capacitance, and (D) relationship between pump current density and cell size (capacitance). The exponential decay curve was fitted to all points using the equation Y = (Y₀− Plateau) × exp(−K × X) + Plateau, where K is the ‘rate’ constant. (E) LVDP over a 20 min period of aerobic perfusion (pacing rate 550 bpm). (F) LV pressure–frequency relationship in sham and banded hearts. [Numerical data are mean ± SEM, A and C, 23 cells from eight sham mice (n = 8) and 27 cells from nine banded mice (n = 9). In E and F, comparison between groups (sham and banded, n = 6/group) was performed using two-way ANOVA, repeated measures, *P < 0.05].

Figure 4

Basal protein expression and PLM phosphorylation measured in hearts from WT and PLM^3SA mice. Representative western blots for Na/K pump α subunits, NCX and SERCA 2a proteins (A) and PLM phosphorylation (B). Quantitative changes in protein expression (C–E). Alpha1/2, Na/K ATPase α1/2 subunits; PLM, phospholemman; SERCA2a, sarco/endoplasmic reticulum Ca ATPase type 2a; NCX, sodium–calcium exchanger. WT and PLM^3SA samples were run on the same gels. In A and B, each pair of rows represents images cropped from different gels probed with different antibodies as shown. CSQ was used as a loading control. Data are mean ± SEM, *P < 0.05; n = 10–12 (WT and PLM^3SA).

Figure 5

Cardiac function and morphometry assessed by echocardiography in sham and banded WT and PLM^3SA mice. (A) Representative echocardiographic images (M-mode) 8 weeks after aorta constriction. (B) Quantitative measures of cardiac function and hypertrophy in sham and banded hearts. LV/Bw is left ventricular weight as a function of body weight. Data are mean ± SEM, P < 0.05; n = 10 (sham, WT), n = 9 (banded, WT), n = 6 (sham, PLM^3SA), n = 7 (banded, PLM^3SA). Comparison was performed using two-way ANOVA (*P < 0.05).

Figure 6

Na/K pump current and intracellular Na measurements in myocytes isolated from sham and banded WT and PLM^3SA hearts. (A) Cell capacitance showing significantly increased myocyte hypertrophy in the PLM^3SA banded group, (B) mean pump current density in cells isolated from sham and banded WT and PLM^3SA hearts normalized to cell capacitance, and (C) relationship between pump current density and cell size (capacitance). Each individual dataset is fitted with a linear regression. (D) Example trace showing calibration of SBFI fluorescence, and (E) mean intracellular Na concentrations in sham and banded myocytes from WT and PLM^3SA mice. Data in A, B, and E are mean ± SEM; in A and B, seven cells from four sham WT (n = 4), nine cells from five banded WT (n = 5), four cells from four sham PLM^3SA (n = 4), and 10 cells from five banded PLM^3SA hearts (n = 5); in E, 15 cells from five sham WT (n = 5), 12 cells from seven banded WT (n = 7), 24 cells from seven sham PLM^3SA (n = 7), and 27 cells from five banded PLM^3SA hearts (n = 5). Comparison was performed using two-way ANOVA (*P < 0.05).

Figure 7

Intracellular Na and NCX activity measurements in paced myocytes isolated from WT and PLM^3SA hearts. (A) Mean intracellular Na concentrations in myocytes from WT and PLM^3SA mice paced at 1 Hz, (B) example trace showing Ca transients (0.5 Hz) and the caffeine-induced Ca release from the SR used to calculate the NCX activity, (C) original traces of caffeine-induced Ca transient decay in myocytes isolated from WT and PLM^3SA hearts (a mono-exponential function was fitted to the curve the time-constant of which is assumed to be largely inversely related to NCX activity), and (D) forward-mode NCX activity assessed by the mean time-constants of the caffeine-induced Ca transient decay in cells isolated from WT and PLM^3SA mice. Data in A and D are mean ± SEM; in A, 46 cells from three WT hearts (n = 3) and 34 cells from three PLM^3SA hearts (n = 3); in D, 29 cells from five WT hearts (n = 5) and 30 cells from five PLM^3SA hearts (n = 5), *P < 0.05.