Original article
A SERCA2 pump with an increased Ca2+ affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span

https://doi.org/10.1016/j.yjmcc.2006.05.014Get rights and content

Abstract

Abnormal Ca2+ cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca2+ pump, the sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca2+ by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca2+ affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB−/− mice. The affinity for Ca2+ of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca2+-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca2+ is physiologically important for normal cardiac function. An improved Ca2+ transport in the sarcoplasmic reticulum may correct Ca2+ mishandling in heart failure, but a SERCA pump with a much higher Ca2+ affinity may be detrimental.

Introduction

Uptake of Ca2+ into the sarcoplasmic reticulum (SR) through ATP-dependent Ca2+ pumps (SERCA) critically determines the rate of cardiac muscle relaxation [1]. SERCA, mainly represented by the SERCA2a isoform in the heart [2], also determines the Ca2+ loading of the SR and therefore the amount of Ca2+ release and cardiac contractility [1]. In the ventricles, SERCA's Ca2+ affinity and activity are modulated via its inhibitor phospholamban (PLB). Beta-adrenergic stimulation phosphorylates and inactivates PLB, increasing SERCA's Ca2+ affinity, and promoting cardiac relaxation and contractility [3].

Lowering the Ca2+ affinity of SERCA or reducing the Ca2+ load of the SR e.g. via increased PLB inhibition or decreased SERCA levels can cause heart failure in both mice and humans [3], [4]. Indeed, enforcing fourfold overexpression of PLB in mice ultimately resulted in heart failure [4]. Humans expressing a phosphorylation-defective PLB mutant (Arg-9-Cys) also developed juvenile heart failure due to chronic SERCA inhibition [5].

In contrast, increasing SR Ca2+ uptake in general enhances cardiac function [3], [6] and may therefore favorably affect the course of heart failure [6]. In rodent models of heart failure, cardiac dysfunction was effectively alleviated by augmenting the Ca2+ affinity of the SERCA pump via gene inactivation or antisense-mediated downregulation of PLB [3], [7], [8], [9]. However, loss of PLB failed to rescue cardiac dysfunction in two other genetic mouse models of familial hypertrophic cardiomyopathy [10]. Moreover, gene profiling in heart failure patients revealed a clearly positive correlation between the development of juvenile dilated cardiomyopathy and the expression of a truncated PLB (Leu-39-stop) incapable of inhibiting SERCA [11]. Taken together, increasing the Ca2+ affinity of the pump might not always be beneficial for cardiac dysfunction. We recently generated gene-targeted mice (SERCA2b/b or SKO; [12]) in which SERCA2a was replaced by the SERCA2b isoform with a higher affinity for Ca2+ [13]. SERCA2b is normally only expressed at low levels in the heart (2% of total SERCA content) [14]. These SKO mice suffered from non-pressure overload concentric left ventricular (LV) hypertrophy and moderate cardiac dysfunction. Though the Ca2+ affinity of the pump was increased, SERCA levels dropped by a factor of two and PLB levels were doubled [12]. Moreover, PLB phosphorylation was also reduced in SKO (the ratio of PLB phosphorylated at Ser16 was twofold lower, but phosphorylation was unaltered at Thr17; Vangheluwe, unpublished data). Together, these observations suggest increased SERCA inhibition by PLB. We hypothesized that the enhanced PLB inhibition contributes to the observed cardiac dysfunction and remodeling in SKO which predicts an improved cardiac function upon removal of PLB (as in [3], [7], [8], [9]). Alternatively, the increased PLB levels could be compensatory and protect the SKO heart by reducing the high Ca2+ affinity of SERCA2b. To test these hypotheses, we crossed SKO with PLB−/− mice and explored the consequences on cardiac function and remodeling of the resulting DKO mice.

Section snippets

Materials and methods

Genotyping of the SERCA2b/b (129sV/Swiss) × PLB−/− (129sV/FVBN) cross was performed as previously described [12], [15]. Experiments were conducted with adult mice (10–16 weeks) of either sex, according to the Guide for the Care and Use of Laboratory Animals (NIH).

Ventricular homogenates were analyzed by standard Western blotting [12]. Phosphatase activity was inhibited with (mmol/L) 25 NaF, 5 Na-EDTA, 5 Na4P2O7 and a phosphatase inhibitor cocktail 1 (Sigma, P2850). Primary antibodies against PLB

PLB compensates for SERCA2b’s high Ca2+ affinity in SKO mice

To examine the functional impact of the PLB upregulation on the apparent affinity of the SR pump for Ca2+, SKO mice were intercrossed with mice lacking PLB (PKO). Ventricular microsomes of the double-mutants (DKO) were prepared and the Ca2+-dependent ATPase activity was measured, from which the Km as a measure of SR Ca2+-pump affinity was calculated. In DKO mice, the Km was 4.8-fold lower than in SKO (Fig. 1A). Thus, increased expression of PLB functionally compensated for the increased Ca2+

Discussion

The work presented here provides unequivocal evidence that a SERCA pump with a too high Ca2+ affinity can elicit cardiac hypertrophy and reduce life span, and that PLB upregulation protects against a pump with a very high affinity for Ca2+. Cardiac hypertrophy was associated with preserved contractility, but baseline diastolic dysfunction and a high risk of cardiac death during beta-adrenergic stimulation. Interestingly, our findings in DKO mice (which lack PLB) mirror the cardiac consequences

Acknowledgments

P.V. is a postdoctoral fellow of the Research Fund K.U. Leuven. M.T. is an IWT research fellow. This work was supported by the Interuniversity Attraction Poles Programme-Belgian Science Policy P5/05 and by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen G.0166.04 and NIH HL26057. This work has been supported by EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart.

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