Dataset Information

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase.

ABSTRACT: 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.

SUBMITTER: Ursitti JA

PROVIDER: S-EPMC1983066 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase.

Ursitti Jeanine A JA Petrich Brian G BG Lee Pervis C PC Resneck Wendy G WG Ye Xin X Yang Jay J Randall William R WR Bloch Robert J RJ Wang Yibin Y

Journal of molecular and cellular cardiology 20070205 3

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 ...[more]

PMID: 17276456

Similar Datasets

Project description: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.

Dataset Information

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase.

Publications

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets