Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice

Abstract

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca(2+) was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca(2+) transients and peak tensions were reduced, whereas decay times of Ca(2+) transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to beta-adrenergic agonists may prevent progression to heart failure.

Fig. 1.

Strategy for generation of TG mice with cardiac–specific overexpression of NF-SLN. (A) HPRT targeting construct. The targeting vector contains the promoter and the first two exons of Hprt, the α-myosin heavy-chain promoter, NF-SLN, and the human growth factor (HGF) polyadenylation signal sequence. (B) Depleted Hprt. The mutant Hprt gene in the HPRT-deficient E14Tg2a embryonic stem cell line, BK4, is shown with the sites of targeting vector insertion. (C) Transfection of this vector into E14Tg2a cells, together with successful homologous recombination, corrects the HPRT deficiency, restoring the ability of the cells to grow in hypoxanthine/aminopterin/thymidine medium, allowing for rapid screening of positive clones. (D) PCR genotyping was performed by using a 5′ primer within the α-MHC promoter sequence and a 3′ primer within the NF-SLN sequence (forward and reverse arrows in C). Neg., negative control. (E) Protein expression of NF-SLN. The expression levels of NF-SLN were determined in various tissues by Western blot analysis with the anti-FLAG antibody, M2.

Fig. 2.

Relative quantification of NF-SLN expression in microsomal fractions from transfected HEK-293 cells and TG hearts. HEK-293 cells were grown to confluence in a 100-cm plate and transfected with 6 μgofPLN,6 μg of NF-SLN, and 8 μg of SERCA1a cDNAs. (A) After 48 h, microsomal fractions were isolated, and 7.5 μg of total protein was separated by SDS/PAGE and stained separately with the M2 antibody against NF-SLN and the 1D11 antibody against PLN. The ratio of NF-SLN band intensity to PLN band intensity averaged 95% (n = 3). Microsomal fractions were also isolated from hearts from 10-week-old TG mice, and Ca²⁺ transport activity was shown to be superinhibited (data not shown). Microsomal proteins (12.5 μg) were separated by SDS/PAGE and stained separately with antibody M2 against NF-SLN and with antibody 1D11 against PLN. The ratio of NF-SLN band intensity to PLN band intensity averaged 106% (n = 3). In the bottom row, identical aliquots were stained with the antibody 2A7-A1 against SERCA2a. The fact that the band intensity in each of the four lanes varied between 100% and 111% shows that SERCA2a content was comparable in all four samples. (B) Ca²⁺ uptake activity in cardiac ventricular homogenates prepared from TG (n = 3) or wt (n = 3) mice. Data are expressed as mean ± SEM.

Fig. 3.

Ca²⁺ transients and tension in left ventricular papillary muscles. Dissected papillary muscles (n = 8 for TG mice and 10 for wt mice) were microinjected with aequorin. (A and B) Aequorin light signals and tensions (stimulation frequency, 0.2 Hz) were recorded simultaneously at 30°C. Shown are traces representing light signal (A) and tension (B). The light signals were converted to [Ca²⁺]_i by using an in vitro calibration curve. Decay time is the time for the light signal to decay from 75% to 25% of the peak. Relaxation time is the time required for the tension to decay from 75% to 25% of the peak. Time-to-peak light (TPL) and time-to-peak tension (TPT) are the times measured from the onset of stimulus to the peaks of light and tension, respectively. Width and length are those of the isolated papillary muscle preparation. ^*, P < 0.05 vs. wt.

Fig. 4.

Properties of PLN in NF-SLN TG mice. (A) Western blot analyses of ventricular samples to detect PLN and phospho-PLN (285-pPLN) and PLN from wt and TG hearts. Numbers represent the mean ± SEM in arbitrary densitometric units. Note the reduced level of PLN phosphorylation in TG samples. (B) Monomer/pentamer ratio (m/p) of PLN in TG hearts. Boiled or nonboiled microsomal fractions were subjected to immunoblotting with anti-PLN antibody. p, Pentameric PLN; m, monomeric PLN. (C) Physical interaction of PLN with SERCA2a in TG hearts. Microsomal fractions were subjected to immunoprecipitation with anti-PLN antibody, 1D11, or anti-FLAG antibody, M2. Precipitates were separated by SDS/PAGE, and SERCA2a or PLN were detected by immunoblotting.

Fig. 5.

Response to isoproterenol. (A) Isolated papillary muscles from wt (n = 7) and TG (n = 9) mice were isolated and analyzed as described in the legend to Fig. 3 in the absence (CTL) and presence (Iso) of 100 nM isoproterenol applied to the surface of the muscles. (B) Hemodynamic measurements on whole hearts were taken after the administration of 2 μg/g isoproterenol through the jugular vein of anesthetized animals. Under resting conditions (CTL) and at the point of maximal changes as assessed by continuous hemodynamic recordings (Iso), ventricular tissue (n = 3 in each condition) was isolated, snap-frozen, and analyzed by immunoblotting with antibodies to PLN (1D11) and phosphorylated PLN (285). Numbers represent the mean ± SEM in arbitrary densitometric units. p, Pentameric PLN; m, monomeric PLN; m/p, the ratio of monomeric to pentameric PLN.