MOLECULAR AND CELLULAR BIOLOGY, 0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4119–4128.2001 July 2001, p. 4119–4128 Vol. 21, No. 13 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Loss of Annexin A7 Leads to Alterations in Frequency-Induced Shortening of Isolated Murine Cardiomyocytes CLAUDIA HERR, 1 NEIL SMYTH, 2 SUSANNE ULLRICH, 3 FAN YUN, 3 PHILLIP SASSE, 3 JU ¨ RGEN HESCHELER, 3 BERND FLEISCHMANN, 3 KATRIN LASEK, 4 KLARA BRIXIUS, 4 ROBERT H. G. SCHWINGER, 4 REINHARD FA ¨ SSLER, 5 ROLF SCHRO ¨ DER, 6 AND ANGELIKA A. NOEGEL 1 * Institute of Biochemistry I 1 and II, 2 Department of Neurophysiology, 3 and Laboratory of Muscle Research and Molecular Cardiology, Clinic III of Internal Medicine, 4 University of Cologne, 50931 Cologne, and Department of Neurology, University Hospital Bonn, Bonn, 6 Germany, and Department of Experimental Pathology, Lund University, Lund, Sweden 5 Received 21 December 2000/Returned for modification 1 February 2001/Accepted 6 April 2001 Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca 2 channel and as Ca 2 -activated GTPase, thus inducing Ca 2 /GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca 2 -induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca 2 regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca 2 homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7 / mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca 2 homoeostasis. Annexins are a family of Ca 2+ -and phospholipid-binding proteins encoded by at least 12 different genes in mammals and by numerous other genes in invertebrates and plants. They are characterized by a bipartite structure with a variable N-termi- nal domain and a conserved C-terminal core. The latter is formed by either four- or eightfold repeats of approximately 70 amino acids, each repeat carrying a Ca 2+ -binding site. This C-terminal domain is also responsible for phospholipid bind- ing. The unique N-terminal regions are thought to confer func- tional diversity (35). Although annexins have been well char- acterized structurally and biochemically, their cellular importance is unclear. Several roles have been proposed, such as the inhibition of phospholipase A2 and of blood coagulation (36, 48), the aggregation of chromaffin granules (10), cross- linking functions in the cell cortex (13), endo- and exocytosis (1, 11), as well as functioning in the regulation and formation of ion channels (18). Annexin A7 (also called synexin), the first family member to be described, was isolated as the agent that mediated aggre- gation of chromaffin granules and fusion of membranes and phospholipids (8). Annexin A7 is unusual in that it carries a long N-terminal extension of more than 100 amino acids. Al- ternative splicing may lead to the inclusion of an extra exon in this region and leads to the generation of two isoforms of 47 and 51 kDa. The 47-kDa protein is present in all tissues except for skeletal muscle. Here the 47-kDa form is lost upon myo- blast differentiation, with the 51-kDa isoform being exclusively present in myotubes (6, 38). Both forms are expressed in the heart and brain (24, 38). As with other family members, the function of annexin A7 remains unclear. There are reports of it acting as a Ca 2+ channel and as Ca 2+ -activated GTPase, supporting Ca 2+ / GTP-dependent secretion (5). Annexin A7 is found in the vicinity of secretory vesicles, on subcellular membranous struc- tures, and on plasma membranes (6, 22), suggesting a possible role in Ca 2+ -mediated exocytosis. However, in spite of these properties, it has been difficult to unambiguously establish its function. Srivastava et al. recently described an annexin A7 knockout mouse in which its absence resulted in lethality at embryonic day (E10) due to cerebral hemorrhaging; further, heterozygous mice had defects in inositol 1,4,5-triphosphate (IP 3 ) receptor expression, Ca 2+ signaling, and a lowered insu- lin content in the endocrine pancreas (39). To describe further its in vivo function, annexin A7-deficient (Anxa7 -/- ) mice were generated by homologous recombina- tion in embryonic stem (ES) cells. The resulting Anxa7 -/- mice are viable, are fertile, and exhibit no differences from wild-type (WT) animals with respect to insulin production and secretion. However, while no abnormalities in Ca 2+ homeosta- sis are seen at the early embryonic stage, adult mice display defects in cardiomyocyte function. MATERIALS AND METHODS Construction of an Anxa7 targeting construct. An EMBL3 mouse genomic library of the 129SV mouse line (46) was screened with a full-length mouse Anxa7 cDNA as a probe. A 15-kb genomic fragment, containing exons 4 to 13, was used to generate the targeting vector in pBluescript SK. A MunI site in intron 4 was deleted by partial digestion and religation. The sequences were then interrupted at the remaining MunI site in exon 8 by insertion of the neomycin resistance (neo) cassette from plasmid pPNT, resulting in the targeting vector * Corresponding author. Mailing address: Institute of Biochemistry I, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany. Phone: 49 221 478 6980. Fax: 49 221 478 6979. E-mail: noegel@uni-koeln.de. 4119