Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c)

Abstract

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Figure 1. HEK-293 cells stably transfected with SERCA2a, SERCA2b and SERCA2c cDNA constructs overexpress the corresponding recombinant proteins

(A) Comparison of the stably transfected SERCA2 proteins at mRNA level. RT reactions (n=5) were carried out starting from 250 ng of template mRNA. Subsequent PCR reactions (n=5) were performed for GAPDH and SERCA2b for 19 cycles, and for SERCA2a and SERCA2c for 20 cycles. No DNA contamination was observed when SERCA2b mRNA amplification was performed in the absence of reverse-transcribed template (-RT). The numbers indicate the sizes of PCR products in base pairs. (B) C-termini of SERCA2 proteins showing the epitopes (underlined or boxed sequences) for SERCA2-specific polyclonal antibodies [6,20]. ‘Solidus’ marks the splice sites. The boxed amino acid sequences represent the two peptides (P1 and P2) used for SERCA2c-specific immunization. (C) Comparison of the stably transfected SERCA2 proteins at protein level. Membrane proteins were isolated from HEK-293 cells transfected with empty vector pcDNA3.1, SERCA2a, SERCA2b and SERCA2c cDNAs. Microsomes (10 μg) were used for Western blotting using the indicated antibodies. To test the specificity of the anti-SERCA2c antibody, the blots were treated either in the absence or presence of 10 μM peptide (P1) (n=3). The numbers indicate protein molecular masses (in kDa).

Figure 2. Recombinant SERCA2a, SERCA2b and SERCA2c proteins modulate [Ca²⁺]_ER

Measurements of Ca²⁺ levels have been performed in the cytosol of HEK-293 cells transfected with empty vector pcDNA3.1, SERCA2a, SERCA2b and SERCA2c cDNAs as described in [9,11]. (A) [Ca²⁺]_C was recorded in the absence and presence of 2 mM EGTA, and the Ca²⁺ response evoked by 5 μM ionomycin was taken as an estimate of [Ca²⁺]_ER. Arrows show the [Ca²⁺]_C and [Ca²⁺]_ER measurements. The double-headed arrow shows the [Ca²⁺] differences used for calculations. (B) Quantitative comparison of the [Ca²⁺]_C (white bars) and [Ca²⁺]_ER (black bars) of the SERCA2 recombinants (means±S.E.M. for n=9). *P, **P and ***P<0.01 compared with pcDNA3.1, SERCA2a and SERCA2b transfectants respectively.

Figure 3. Recombinant SERCA2c protein shows similar turnover rate to SERCA2b but a lower affinity for Ca²⁺

(A) The ATPase turnover rates were determined as described in [23]. Bars represent the ATPase activities of SERCA2a (70 s⁻¹), SERCA2b (35 s⁻¹) and SERCA2c (36 s⁻¹) compared with SERCA1a (130 s⁻¹) (n=3). (B) Ca²⁺ affinity for activation of phosphorylation from ATP. Phosphorylation was carried out in the presence of 5 μM [γ-³²P]ATP and the indicated concentrations of free Ca²⁺ [23]. The data were normalized by taking as 100% the maximum phosphorylation reached, and the ‘lines’ show the best fits of the Hill equation, giving the following K_0.5 values: SERCA1a, 1.030; SERCA2a, 0.985; SERCA2b, 0.508; and SERCA2c, 1.604 (n=3 or 4).

Figure 4. Differential distribution of SERCA2c mRNA in human tissues

(A) PCRs showing the expression of SERCA2a, SERCA2b, SERCA2c and GAPDH transcripts in the indicated tissues [9,11] were performed using 1.25 ng of normalized cDNAs (Clontech) and the primers indicated in Supplementary Table 1 (at http://www.BiochemJ.org/bj/395/bj3950249add.htm) for 19 cycles (SERCA2a and SERCA2b) and 26 cycles (SERCA2c and GAPDH). (B) For quantifications, the values of heart (normalized to GAPDH) were arbitrarily taken as 100% for SERCA2a, SERCA2c and GAPDH or those of liver for SERCA2b. The expressions are given as percentages of heart or liver values (means±S.E.M. for n=6).

Figure 5. SERCA2c is endogenously expressed in the human heart in a more restricted area than SERCA2a and SERCA2b proteins

(A) Recombinant SERCA2c-containing microsomes (10 μg), total human heart protein lysate from Clontech (200 μg) and protein lysates (C1–C3) isolated from the left ventricle of three non-failing hearts (30 μg) were analysed by Western blotting using the anti-SERCA2c antibody in the absence (anti-SERCA2c) or presence (anti-SERCA2c+P1) of 10 μM peptide (P1) used for immunization (n=3). (B) Co-immunolabelling of α-actinin and SERCA2 proteins in left ventricle sections from human heart. Overview of fixed heart tissue, double stained with anti-α-actinin (A, D and G) and anti-SERCA2a (B), -SERCA2b (E) or -SERCA2c (H) antibodies. C, F and I show merged images of stainings for α-actinin and SERCA2 proteins. Arrows 1, 2 and 3 indicate longitudinal SR, transversal SR and intercalated discs respectively. The scale bar represents 2 μm (n=3).

Figure 6. mRNA and protein expression levels of SERCA2 and PMCAs in normal and failing hearts (representative experiments)

(A) RNA study. The RT–PCR experiment shows the expression of GAPDH (after 19 PCR cycles), SERCA2a (after 16 PCR cycles), SERCA2b (after 19 PCR cycles), SERCA2c and PMCA1c–PMCA1b (after 26 PCR cycles) and PMCA4a–PMCA4b (after 27 PCR cycles) mRNAs in hearts isolated from four normal subjects (C1–C4) and five patients with cardiomyopathies (P1–P5), mixed cardiomyopathy for patient P1 and idiopathic dilated cardiomyopathy for patients P2–P5 using the primers indicated in Supplementary Table 1 (at http://www.BiochemJ.org/bj/395/bj3950249add.htm). (B) Protein study. Western blots of calreticulin (CRT), total SERCA2, SERCA2a, SERCA2c, total PMCAs and PMCA4b proteins using 30 μg of left ventricle lysate proteins and the corresponding anti-calreticulin, IID8, anti-SERCA2a, anti-SERCA2c, 5F10 and JA3 antibodies respectively.

Figure 7. Comparative expression of SERCA2 and PMCA mRNAs and proteins in normal (C1–C4) and failing hearts (P₁–P₅) (summary of densitometric data)

(A) RNA study. For quantifications of non-failing and failing hearts, the mean value of the different normal hearts was arbitrarily taken as 100%. For non-failing hearts, results are expressed as the means±S.E.M. for n=5. For quantifications of failing hearts, results were corrected for GAPDH and are expressed as the means±S.E.M. for n=9–15. *P<0.01 and ^#P<0.05 compared with control. (B) Protein study. For quantifications of non-failing and failing hearts, the mean value of the different non-failing hearts was arbitrarily taken as 100%. For quantifications of failing hearts, results were corrected for calreticulin and are expressed as the means±S.E.M. for n=5–9. *P<0.01 and ^#P<0.05 compared with control.