Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species

Abstract

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

Figure 1. Determination of SLN/SERCA and PLB/SERCA mRNA ratios using RT-PCR

The amount of cDNA used for amplification was adjusted to yield equal amounts of SERCA. The panels in (I) show representative examples of RT-PCR on cDNA of different muscle types (V, ventricles; A, atria; S, soleus; E, EDL). Four different species, as indicated, were analysed in parallel. The primers and PCR conditions summarized in Supplementary Table A (http://www.BiochemJ.org/bj/389/bj3890151add.htm) were used to determine expression levels of SLN, PLB and SERCA. The panels in (II) summarize the PLB/SERCA (grey) or SLN/SERCA (white) mRNA ratio represented as mean percentage (±S.E.M.) compared with atrial levels. For mouse, tissue was pooled from six different animals to prepare total RNA (n=2). For other species, n=3. #SLN/SERCA mRNA ratio significantly different (P<0.05) compared with atria. *PLB/SERCA mRNA ratio significantly different (P<0.05) compared with atria. VENTR, ventricles.

Figure 2. Determination of SERCA isoforms using ratio PCR

Mouse (A), rat (B), rabbit (C) and pig (D) were analysed in parallel. The amount of cDNA used for RT was adjusted to yield equal amounts of total SERCA. Following PCR, restriction digestion (see enzymes in Supplementary Table A at http://www.BiochemJ.org/bj/389/bj3890151add.htm) was performed to discriminate between SERCA1 and SERCA2. The panels in (I) show representative examples of ratio PCRs on cDNA of different muscle types (V, ventricles; A, atria; S, soleus; E, EDL). The panels in (II) summarize the data expressed as the means (±S.E.M.) of percentage SERCA1/total SERCA (grey) or percentage SERCA2/total SERCA (white) mRNA ratios. Mouse tissue was pooled from six different animals to prepare total RNA (n=2). For other species, n=3. VENTR, ventricles.

Figure 3. Characterization of the anti-SLN antibody

(A) Purified SLN (0.1 μg) was visualized with Sypro Orange after SDS/PAGE (lane I). After transfer on to a PVDF membrane, the SLNAP78 antibody recognizes the purified SLN (lane III). No PLB is present in the purified SLN (lane II) as shown by incubation of the blot with an anti-PLB antibody. (B) Addition of antigenic peptide in solution (+) during primary antibody incubation (lane V) can reduce the antibody reaction with purified SLN (−) (lane IV). (C) The SLNAP78 antibody cross-reacts with PLB. Homogenate of mouse atria (70 μg) was transferred to a blot and stained in parallel with the SLNAP78 (lane VI) and anti-PLB antibody (lane VII). PLB (apparent molecular mass 5 kDa) and SLN (apparent molecular mass 4 kDa) were discriminated by size. (D) Comparison of the amino acid sequence of homologous parts of PLB, SLN and the epitope used to develop anti-SLN antibodies. Bold and underlined amino acids are identical with the epitope. As indicated by the two rectangles, the C-terminal part of the PLB sequence shows striking similarity to the corresponding mouse and rat SLN sequence, and to the epitope.

Figure 4. Purified SLN (A), PLB (B) and SERCA (C) proteins were evaluated by SDS/PAGE after staining with Sypro Orange

In parallel, SLN, PLB and SERCA were identified via Western blotting (WB) using the SLNAP78, mA1 and TRY2 antibodies respectively. To determine the absolute amount of SLN, PLB and SERCA in the standard, the intensity of the purified protein bands on the Sypro-Orange-stained gel were compared with two protein standards of known concentrations: BSA and GST. On the gels, the amounts of BSA and GST are indicated in μg. Serial dilutions (:3) of SLN, PLB and SERCA were analysed.

Figure 5. Western-blot analysis of total homogenates of different muscle types (V, ventricles; A, atria; S, soleus; E, EDL) of (A) mouse, (B) rat, (C) rabbit and (D) pig

The amount of protein on the gel was adjusted to yield equal amounts of SERCA on a Western blot stained with TRY2 anti-SERCA antibody (I). Anti-SLN (II) and anti-PLB antibody (III) were used to stain parallel Western blots loaded with the same relative protein ratios. Cross-reaction of the anti-SLN antibody with PLB is indicated in (II). To estimate the amount of SLN, PLB and SERCA, samples were compared with an absolute standard for SLN, PLB and SERCA. Results are presented in Table 3. Mouse tissue was pooled from six different animals to prepare homogenates (n=2). For other species, n=3.

Figure 6. Representative examples of the relative quantification of SLN mRNA and protein expression in atria or ventricle of PLB^−/− and SERCA2^b/b

RPA was performed to estimate SLN mRNA levels in atria or ventricle of PLB^−/− (A) and SERCA2^b/b (B). (C) After Western blotting (WB) of 60 μg homogenates, the SLNAP78 antibody was used to determine relative SLN protein levels in atria of PLB^−/− and SERCA2^b/b. In parallel, blots were stained with the anti-PLB antibody mA1. (D) Summary of the SLN mRNA, SLN protein and PLB protein expression levels. Atria were pooled from six to eight different animals to prepare total RNA or homogenates. Analysis was performed in triplicate. Results are mean percentages±S.E.M. of expression compared with WT. *Significantly different compared with WT (P<0.05).