The human phospholamban Arg14-deletion mutant localizes to plasma membrane and interacts with the Na/K-ATPase

Abstract

Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.

Fig. 1

Quantitative immunoblotting of PLN and SERCA2a in mouse cardiac homogenates and SR Ca uptake in transgenic and wild type hearts. (A): Cardiac homogenates were subjected to 13% SDS-polyacrylamide gel electrophoresis and electroblotted on nitrocellulose membranes. The membranes were probed with a PLN antibody or SERCA antibody. PLNp, PLN pentamer; PLNm, PLN monomer. (B): Expression levels of phosphorylated PLN protein at Ser¹⁶ and Thr¹⁷ in PLN-R14Del relative to PLN-WT hearts. (C): Quantification of total PLN protein in PLN-R14Del (Lines 1 and 2) relative to PLN-WT hearts. Data represent the mean ± SEM. A minimum of 4 hearts were analyzed for each genotype or line. (D): Initial rates of SR Ca uptake in cardiac homogenates from PLN-WT (●, n = 4), PLN-KO (■, n = 4) and PLN-R14Del (▲, n = 4) mice. The points represent the mean ± SEM of four experiments (each in triplicate), using individual hearts. The data were analyzed by nonlinear regression using Microcal Origin software (version 6.0). Inset shows the EC50 values for PLN-WT (n = 4), PLN-KO (n= 4) and PLN-R14Del (n = 4) hearts. The maximal Ca-uptake velocities (Vmax) were 63.4±3.8 mol/mg/min for PLN-WT, 81.1±9.9 mol/mg/min for PLN-KO and 74.2±10.0 mol/mg/min for PLN-R14Del.

Fig. 2

Mechanical parameters and Ca transients in isolated cardiomyocytes at baseline and under isoproterenol (100nM) stimulation. (A): Rates of shortening (+dL/dt), (B): Rates of relengthening (−dL/dt), (C): Shortening fraction (FS%), and (D): Cell length in isolated cardiomyocytes from wild type mice (PLN-WT, n = 5), PLN-KO (n = 4) and mutant PLN mice (PLN-R14Del, n = 6). Values are mean ± SEM. At least 27 to 80 cells were used for measurements of each parameter. *, p < 0.05 vs PLN-WT. (E): Ca transient amplitude (340/380 nm ratio) obtained from PLN-WT (n = 4), PLN-KO (n = 4) and PLN-R14Del (n = 4). Values are mean ± SEM. A minimum of 42–70 cells were studied. *, p < 0.05 vs PLN-WT. (F): Analysis of caffeine-induced Ca transients in PLN-WT (n = 7–9), PLN-KO (n = 4) and PLN-R14Del (n = 4) cardiomyocytes. 10 mmol/L caffeine was applied and caffeine induced amplitude was assessed. Values are mean ± SEM. A minimum of 30 cells were studied. *, p < 0.05 vs PLN-WT.

Fig. 3

Localization of PLN and SERCA2a proteins in PLN-WT and PLN-R14Del mouse hearts, using immunofluorescence staining. Heart sections from 3 month-old mice were stained with SERCA2a antibody (red) and counterstained with PLN antibody (green). Confocal microscopy was used to assess SERCA2a and PLN localization in wild type (PLN-WT) or mutant PLN (PLN-R14Del) hearts. MERGE indicates the overlay of images. Insets show a higher magnification of the cardiac cell.

Fig. 4

Immunoprecipitations (IP) with either NKA or SERCA antibodies. (A): the NKA antibody was used for immunoprecipitations and blots were probed with NKA (upper panel), PLN (middle panel) and PLM (lower panel) antibodies. All the samples were analyzed on the same gel. (B): upper panel, the SERCA2 specific antibody was used for immunoprecipitation; middle panel, the NKAα1 isoform specific antibody was used as a probe for NKA detection; and lower panel, the PLN specific antibody was used to detect PLN.

Fig. 5

Fluorescence resonance energy transfer (FRET) measurement of PLN-R14Del and NKA interaction. (A): HEK293 cells stably expressing NKAα1-CFP (upper panels) were transfected with YFP-tagged wild-type PLN (PLN-YFP, bottom left) or mutant PLN (PLN-R14Del-YFP, bottom right). (B): Average increase of donor (NKA-CFP) fluorescence after YFP photobleach (normalized to prebleach), indicative of FRET between NKA and PLN-R14Del, but not PLN WT. Controls: Ctl is untransfected and YFP-tagged PLN and phospholemman (PLM) transfected cells were negative and positive controls, respectively. (C): Average increase of donor fluorescence (PLM-CFP) after photobleach (normalized to prebleach), indicative of FRET between PLM-PLM but only minor FRET between PLM-PLN-R14Del. At least 3 separate experiments were performed and values are mean ± SEM, where * indicates p < 0.05.

Fig. 6

Assessment of NKA activity and expression in PLN-WT and PLN-R14Del hearts. (A): Total K⁺ sensitive ATPase activity was increased in PLN-R14Del hearts relative to PLN-WT hearts. n = 8 (B): K⁺ stimulated PNPP assay, indicating significant NKA enzymatic activity in PLN-R14Del hearts. n = 6 (C): Measurement of the NKA expression levels. n = 8 (D): Analysis of sodium calcium exchanger (NCX) expression levels in PLN-WT and PLN-R14Del hearts. n = 4. Values are mean ± SEM. *, p < 0.05 vs PLN-WT.

Fig. 7

Strophanthidin-sensitive NKA activity in PLN-WT and PLN-R14Del hearts. (A): Total ATPase activity was increased in R14del cardiac homogenates under basal conditions or in the absence of an inhibitor. ATPase activities in the presence of strophanthidin, a specific NKA inhibitor, were similar between PLN-WT and PLN-R14Del hearts, suggesting that the increase in basal total ATPase activity was not due to the contribution of other ATPases but an increase in NKA. (B): NKA activity was calculated by subtracting the ATPase activity in the presence of inhibitor from the activity in the absence of inhibitor. The average of NKA activity in PLN-R14Del heart homogenates was expressed as a fold-increase over PLN-WT for each paired sample. n=7. *, p<0.05.

Fig. 8

G ravimetric and histologic analyses of wild type (PLN- WT) and mutant PLN-(PLN-R14Del) mice. (A and B): Heart, lung and body weights were measured from PLN-WT and PLN-R14Del mice. n = 8. Values are mean ± SEM. *, p < 0.05 vs PLN-WT. (C): Images of hearts from PLN-WT and PLN-R14Del mice at 3 months of age. (D): Longitudinal sections of hearts stained with Masson’s trichrome to reveal interstitial tissue fibrosis.