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.