We used degenerate primers for the amino-and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.

Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [Petrich BG, Gong X , Lerner DL , Wang X , Brown JH , Saffitz JE , Wang Y. c-Jun N-terminal kinase activation mediates downregulation of connexin 43 in cardiomyocytes. Circ Res. 91 (2002) 640-647; B.G. Petrich, B.C. Eloff, D.L. Lerner, A. Kovacs, J.E. Saffitz, D.S. Rosenbaum, Y. Wang, Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J. Biol. Chem. 2004;279: 15330-15338]. We examined the membrane cytoskeletal protein, alphaII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of alphaII-spectrin are expressed in the heart, including one, termed alphaII-SH3i, which contains a 20-amino-acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of alphaII-spectrin labeled the sarcolemma, transverse ("t-") tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for alphaII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, alphaII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of alphaII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of alphaII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, overexpression of the SH3i fragment in the context of repeats 9-11 of alphaII-spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the alphaII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates alphaII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.

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.