Characterization and expression of a heart-selective alternatively spliced variant of alpha II-spectrin, cardi+, during development in the rat

Abstract

Spectrin is a large, flexible protein that stabilizes membranes and organizes proteins and lipids into microdomains in intracellular organelles and at the plasma membrane. Alternative splicing occurs in spectrins, but it is not yet clear if these small variations in structure alter spectrin's functions. Three alternative splice sites have been identified previously for alpha II-spectrin. Here we describe a new alternative splice site, a 21-amino acid sequence in the 21st spectrin repeat that is only expressed in significant amounts in cardiac muscle (GenBank GQ502182). The insert, which we term alpha II-cardi+, results in an insertion within the high affinity nucleation site for binding of alpha-spectrins to beta-spectrins. To assess the developmental regulation of the alpha II-cardi+ isoform, we used qRT-PCR and quantitative immunoblotting methods to measure the levels of this form and the alpha II-cardi- form in the cardiac muscles of rats, from embryonic day 16 (E16) through adulthood. The alpha II-cardi+ isoform constituted approximately 26% of the total alpha II-spectrin in E16 hearts but decreased to approximately 6% of the total after 3 weeks of age. We used long-range RT-PCR and Southern blot hybridization to examine possible linkage of the alpha II-cardi+ alternatively spliced sequence with alternatively spliced sequences of alpha II-spectrin that had been previously reported. We identified two new isoforms of alpha II-spectrin containing the cardi+ insert. These were named alpha II Sigma 9 and alpha II Sigma 10 in accordance with the spectrin naming conventions. In vitro studies of recombinant alpha II-spectrin polypeptides representing the two splice variants of alpha II-spectrin, alpha II-cardi+ and alpha II-cardi-, revealed that the alpha II-cardi+ subunit has lower affinity for the complementary site in repeats 1-4 of betaII-spectrin, with a K(D) value of approximately 1 nM, as measured by surface plasmon resonance (SPR). In addition, the alpha II-cardi+ form showed 1.8-fold lower levels of binding to its site on beta II-spectrin than the alpha II-cardi- form, both by SPR and blot overlay. This suggests that the 21-amino acid insert prevented some of the alpha II-cardi+ form from interacting with beta II-spectrin. Fusion proteins expressing the alpha II-cardi+ sequence within the two terminal spectrin repeats of alpha II-spectrin were insoluble in solution and aggregated in neonatal myocytes, consistent with the possibility that this insert removes a significant portion of the protein from the population that can bind beta subunits. Neonatal rat cardiomyocytes infected with adenovirus encoding GFP-fusion proteins of repeats 18-21 of alpha II-spectrin with the cardi+ insert formed many new processes. These processes were only rarely seen in myocytes expressing the fusion protein lacking the insert or in controls expressing only GFP. Our results suggest that the embryonic mammalian heart expresses a significant amount of alpha II-spectrin with a reduced avidity for beta-spectrin and the ability to promote myocyte growth.

Figure 1. An alternatively spliced sequence of 63 nucleotides in spectrin repeat 21, near the C-terminus of cardiac αII-spectrin

This figure diagrams the structure of αII-spectrin with the position of the cardi+ insert in repeat 21 marked. Below the cartoon is the sequence of the 63 nucleotides (in red) present in the cDNA, and the amino acids (in blue) of the cardi+ insert.

Figure 2. The αII-cardi+ splice variant is expressed in rat cardiac muscle

A. RT-PCR was performed on mRNA extracted from brain (Br), kidney (Kd), skeletal muscle (Sk) and cardiac muscle (H) of embryonic day 19 (E19) and 6 month old (M6) rats. The sequence encoding the αII-cardi+ splice variant was detected in brain and heart tissues from E19 rat, but only in heart from M6 rat. B. Immunoblotting with antibodies specific for the αII-cardi+ epitope show that it is expressed in significant amounts in heart tissue. Both RT-PCR and immunoblotting suggests that αII-cardi+ is much more prominent in late embryonic than in adult heart.

Figure 3. The αII-cardi+ splice variant concentrates intracellularly at the level of the Z-line in cardiac muscle, but not skeletal muscle

Enzymatically dissociated adult rat cardiomyocytes and skeletal muscle fibers were fixed and stained with antibodies to all forms of αII-spectrin or specific for the αII-cardi+ epitope. Immunofluorecence shows that general antibodies to αII-spectrin label both the sarcolemma (S, yellow arrow) and structures present at the level of the Z-disks (T-tubule, T, green arrow) in cardiac muscle (A) and skeletal muscle (B), whereas the antibody specific for αII-cardi+ immunolabels only at the level of Z-disks in cardiac muscle (C) and fails to label any distinctive structures in skeletal muscle (D).

Figure 4. Real-time RT-PCR of mRNA encoding αII-cardi− and αII-cardi+

mRNAs were isolated from rat heart tissues at E16, E19, D1, D3, D7, D21 and M6 of age. Quantitative RT-PCR was performed with primers specific for αII-cardi− and αII-cardi+ (see Materials and Methods, Supplementary Table 1). A. mRNA for αII-cardi− (red) and αII-cardi+ (blue), expressed as % total mRNA encoding the two isoforms (n=4). The differences with development were highly significant (p<0.001). B. mRNA encoding αII-cardi+ decreases in amount between E16 and M6, but those encoding αII-cardi− do not (after normalizing 18S RNA, data not shown).

Figure 5. Three alternatively spliced variants of αII-spectrin are expressed independently in rat heart

A. Eight plasmid DNAs containing 3.7 kb of RT-PCR products, amplified by the primers listed in the table 1, were placed onto Nybond-N+ membrane and hybridized to the αII-spectrin probe, which was shared by all variants (A, top row) and showed positive in all clones. The membrane was then stripped and rehybridized with probes specific to the SH3+ (A, middle row) and Cardi+ (A, bottom row) variants. The probes used, and the scores for labeling of each plasmid, are summarized in the table below the blots. For information on the probes used, please see Supplementary Table 1. B. Additional sequencing of all RT-PCR products was performed. These experiments confirmed the results of Southern blots and also showed the presence or absence of the 5 amino acid insert in repeat 15 (insert 2) and the presence of the 6 amino acid insert (insert 3) in repeat 21 in all sequenced clones, which could not be assayed by blotting. The results indicate that the expression of mRNAs encoding the SH3+ insert (insert 1), the 5 amino acid insert in repeat 15 (insert 2), and the cardi+ insert in repeat 21 (cardiac-selective) are not linked. ^*The conventional isoform name is given in column 2, identifying αIIΣ9 and αIIΣ10 as new isoforms of αII-spectrin, according to previously identified isoforms [1].

Figure 5. Three alternatively spliced variants of αII-spectrin are expressed independently in rat heart

A. Eight plasmid DNAs containing 3.7 kb of RT-PCR products, amplified by the primers listed in the table 1, were placed onto Nybond-N+ membrane and hybridized to the αII-spectrin probe, which was shared by all variants (A, top row) and showed positive in all clones. The membrane was then stripped and rehybridized with probes specific to the SH3+ (A, middle row) and Cardi+ (A, bottom row) variants. The probes used, and the scores for labeling of each plasmid, are summarized in the table below the blots. For information on the probes used, please see Supplementary Table 1. B. Additional sequencing of all RT-PCR products was performed. These experiments confirmed the results of Southern blots and also showed the presence or absence of the 5 amino acid insert in repeat 15 (insert 2) and the presence of the 6 amino acid insert (insert 3) in repeat 21 in all sequenced clones, which could not be assayed by blotting. The results indicate that the expression of mRNAs encoding the SH3+ insert (insert 1), the 5 amino acid insert in repeat 15 (insert 2), and the cardi+ insert in repeat 21 (cardiac-selective) are not linked. ^*The conventional isoform name is given in column 2, identifying αIIΣ9 and αIIΣ10 as new isoforms of αII-spectrin, according to previously identified isoforms [1].

Figure 6. Binding of αII-cardi+ to β-spectrins in blot overlay

A. MBP-αII-cardi+ and MBP- αII-cardi− fusion proteins (2 μg each) were separated by SDS-PAGE, blotted with GST-βII-spectrin-1-4 (6.6 nM) and then with antibodies to GST (left) or to MBP (right, loading control). The results reveal that MBP-αII-cardi− binds more avidly than MBP-αII-cardi+ to GST-βII-spectrin-1-4 (6A. left). Similar amounts were present on the blot, as indicated by antibodies to MBP (6A right). B. Quantitations of blot overlays in A (n=4). The differences in binding of the αII-cardi− and αII-cardi+ fusion proteins are significant (*, p<0.05).

Figure 7. Binding kinetics measured by surface plasmon resonance

A. Binding of MBP-αII-cardi+ and MBP-αII-cardi− to GST-βII-1-4 at saturation, in response units. The differences were highly significant (p<0.001, n= 4). B, C. Binding at different concentrations, from 0 to 20 nM, of αII-cardi+ and αII-cardi− fusion proteins was measured (colored lines, from top to bottom, 20 nM, 10 nM, 5 nM, 2.5 nM 1.25 nM and 0) and fit by Michaelis-Menten kinetics (black lines). D-F. Values for binding affinities, and association and dissociation rates for αII-cardi+ and αII-cardi−. The results show that αII-cardi− (KD=0.57 nM) binds to βII-1-4 more avidly than αII-cardi+ (KD=1.09 nM), consistent with results from blot overlays, and that its higher avidity is due to a faster association rate, which leads to tighter binding.

Figure 8. Overexpression of αII-cardi+ in cardiomyocytes promotes cell growth

A, B, C. Neonatal rat cardiomyocytes were infected with adenovirus encoding either GFP-αII-cardi+ (A, green) or GFP-αII-cardi− (B, green) or GFP alone (C, green) and co-labeled with antibodies to α-actinin (A′, A″, B′ B″ and C′ C″ red). Areas of overlapping label are shown in yellow. White arrowhead indicates aggregation (A″, a″) and white arrow shows new processes (A″, B″, C″ and a″, b″, c″). D. Cells were examined for the presence of newly forming processes and aggregates of the GFP fusion proteins, and the numbers were quantitated. Controls included cells that were infected with GFP alone (C, GFP), or not infected (intact. Differences between cells expressing GFP-αII-cardi+ and othercells were highly significant (p<0.001).

Figure 8. Overexpression of αII-cardi+ in cardiomyocytes promotes cell growth

A, B, C. Neonatal rat cardiomyocytes were infected with adenovirus encoding either GFP-αII-cardi+ (A, green) or GFP-αII-cardi− (B, green) or GFP alone (C, green) and co-labeled with antibodies to α-actinin (A′, A″, B′ B″ and C′ C″ red). Areas of overlapping label are shown in yellow. White arrowhead indicates aggregation (A″, a″) and white arrow shows new processes (A″, B″, C″ and a″, b″, c″). D. Cells were examined for the presence of newly forming processes and aggregates of the GFP fusion proteins, and the numbers were quantitated. Controls included cells that were infected with GFP alone (C, GFP), or not infected (intact. Differences between cells expressing GFP-αII-cardi+ and othercells were highly significant (p<0.001).