Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Abstract

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.

Fig. 1.

Targeted disruption of SLN gene. (A) Schematic representation of the SLN gene knockout strategy. Exon 2 was replaced with a neomycin gene in reverse orientation. (B) RT-PCR analysis of SLN mRNA expression in the atria and ventricles of WT and homozygous SLN knockout (sln^−/−) mice. (C) Western blotting analysis of SLN protein expression. SR-enriched microsomal fractions prepared from atria (A, 1 μg), and ventricles (V, 5 μg) of WT and sln^−/− mice were separated on a 16% Tricine PAGE and immunoprobed with anti-rabbit SLN antibody.

Fig. 2.

Quantification of SR Ca²⁺ handling proteins. (A) Two different concentrations of total homogenates (4 and 8 μg for SERCA2a, PLB, and CSQ; 10 and 20 μg for DHPRα, RyR, and triadin) prepared from atrial and ventricular tissues of WT and sln^−/− mice were separated on SDS/PAGE and immunoprobed with specific antibodies. (B) Total homogenates prepared from sln^−/− atria and ventricles were unboiled and analyzed for PLB monomers (PLB_m) and pentamers (PLB_p) by Western blot analysis. To determine the basal phosphorylation of PLB at serine-16 (PLB-S¹⁶) and threonine-17 (PLB-T¹⁷), snap-frozen samples were processed for protein extraction and immunoprobed with S¹⁶- or T¹⁷-specific PLB antibodies. Data shown are representative of three independent experiments.

Fig. 3.

Calcium uptake function in sln^−/− atria and ventricles. Ca²⁺ uptake assays were performed by using total homogenates from atria (A) and ventricles (B) of 24-week-old mice. For each atrial experiment, atria from four mice were pooled. n = 4 for each group. The V_max of Ca²⁺ uptake was obtained at pCa 6.0.

Fig. 4.

Mechanical properties of sln^−/− atria. (A) Effect of extracellular Ca²⁺ and ISO on the isometric force generation in isolated left atria from WT and sln^−/− mice. #, isometric force generation in sln^−/− atria (at 2 mM Ca²⁺) was significantly different from WT atria at all Ca²⁺ concentrations; *, isometric force generation in WT atria at 6 mM Ca²⁺ and ISO stimulation was significantly different from the basal contraction at 2 mM Ca²⁺ (P < 0.05). NS, not significant. Time taken for 50% relaxation (RT₅₀) (B) and 90% relaxation (RT₉₀) (C) was significantly faster in the sln^−/− atria. *, P < 0.05 (significant difference between WT and sln^−/− groups). n = 5.

Fig. 5.

Ca²⁺ transients in atrial and ventricular myocytes isolated from WT and sln^−/− mice. (A) Representative examples of line-scan images in response to ISO stimulation showing kinetics of Ca²⁺_i transients recorded from atrial and ventricular myocytes of sln^−/− mice compared with respective WT littermates. Sample records show a line-scan fluorescence images (Upper) and Ca²⁺_i, measured as F/F_o (Lower). (B) Summary data for the amplitude of the total Ca²⁺ transient (A) obtained at baseline and after ISO stimulation for atrial and ventricular myocytes. n = 28 [WT A (−ISO)], 17 [sln^−/− A (−ISO)], 20 [WT V (−ISO)], 19 [sln^−/− V (−ISO)], 9 [WT A (+ISO)], 13 [sln^−/− A (+ISO)], 17 [WT V (+ISO)], and 32 [sln^−/− V (+ISO)] isolated from five to seven animals per group. (C) Summary data for the fast and slow time constants (τ₁ and τ₂) of the Ca²⁺ transient decay at baseline using two exponential functions.