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

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 αII-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 αII-cardi+, results in an insertion within the high affinity nucleation site for binding of α-spectrins to β-spectrins. To assess the developmental regulation of the αII-cardi+ isoform, we used qRT-PCR and quantitative immunoblotting methods to measure the levels of this form and the αII-cardi− form in the cardiac muscles of rats, from embryonic day 16 (E16) through adulthood. The αII-cardi+ isoform constituted ∼ 26% of the total αII-spectrin in E16 hearts but decreased to ∼ 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 αII-cardi+ alternatively spliced sequence with alternatively spliced sequences of αII-spectrin that had been previously reported. We identified two new isoforms of αII-spectrin containing the cardi+ insert. These were named αIIΣ9 and αIIΣ10 in accordance with the spectrin naming conventions. In vitro studies of recombinant αII-spectrin polypeptides representing the two splice variants of αII-spectrin, αII-cardi+ and αII-cardi−, revealed that the αII-cardi+ subunit has lower affinity for the complementary site in repeats 1-4 of βII-spectrin, with a KD value of ∼ 1 nM, as measured by surface plasmon resonance (SPR). In addition, the αII-cardi+ form showed 1.8-fold lower levels of binding to its site on βII-spectrin than the αII-cardi− form, both by SPR and blot overlay. This suggests that the 21-amino acid insert prevented some of the αII-cardi+ form from interacting with βII-spectrin. Fusion proteins expressing the αII-cardi+ sequence within the two terminal spectrin repeats of αII-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 β subunits. Neonatal rat cardiomyocytes infected with adenovirus encoding GFP-fusion proteins of repeats 18-21 of αII-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 αII-spectrin with a reduced avidity for β-spectrin and the ability to promote myocyte growth.