Julia von Maltzahn

Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.

Connexin Expression and Functional Analysis of Gap Junctional Communication in Mouse Embryonic Stem Cells

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43‐deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin‐deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.

Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

The novel mouse connexin39 gene is expressed in developing striated muscle fibers

The recently identified mouse connexin39 (mCx39) gene encodes a peptide of 364 amino acids that shows only 61% sequence similarity to its putative human orthologue connexin40.1 (hCx40.1). The coding regions of mCx39 and hCx40.1 are located on two different exons as described for murine and human connexin36. Northern blot and RT-PCR analyses revealed that mCx39 is expressed after embryonic day (ED) 13.5 up to birth and is absent from the adult stage. Polyclonal antibodies raised to a peptide corresponding to the 16 C-terminal amino acid residues detected a protein band of about 40 kDa apparent molecular mass in lysates of several embryonic tissues. In sections of ED14.5, ED16.5 and neonatal (P0) tissues, immunofluorescent signals were prominent between myotubes in the developing diaphragm, within the intercostal muscle, in the region around the occipital bone, as well as in muscles of the limb, tongue and connective tissue around the eye. These antibodies yielded punctate signals on apposed plasma membranes of HeLa cells transfected with Cx39 cDNA but did not react with wild-type cells. Furthermore, no intercellular permeation of microinjected neurobiotin and other tracers could be detected in Cx39 transfected HeLa cells. However, after microinjection of Alexa488 into myotubes of dissected neonatal diaphragm, we found spreading of this dye into neighbouring cells. As expression of no other known connexin could be verified in these cells, intercellular dye transfer might result from functional expression of Cx39 in developing striated muscle fibers.

Subcellular distribution of connexin45 in OFF bipolar cells of the mouse retina.

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild‐type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45‐EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons. J. Comp. Neurol. 519:433–450, 2011.

Connexin45 Is Expressed in Vascular Smooth Muscle but Its Function Remains Elusive

Connexins (Cx) form gap junctions and allow the coordination of cellular behaviour. In vessels, expression of Cx40, Cx37, and Cx43 is well established and specifically Cx40 serves important functions in endothelial cells. In contrast, expression and physiological functions of Cx45 is unclear although its expression has been suggested in vascular smooth muscle (VSM). Therefore, we studied expression and function of Cx45 in vessels using different mice models allowing to identify and delete Cx45. Smooth muscle cell (SMC)-specific deletion was achieved by the Cre/loxP system using Cre-recombinase driven by a Nestin promoter. Deletion of Cx45 leads concomitantly to the expression of enhanced green fluorescence protein (EGFP) in these mice. Conduction of vasomotor responses was studied in cremasteric arterioles using intravital microscopy and arterial pressure was measured telemetrically. Cx45 is transcriptionally expressed in VSM as detected by EGFP expression in SMC-specific Cx45-deficient mice (Cx45fl/fl:Nestin-Cre) but not in endothelial cells (Cx45fl/fl:TIE2-Cre). Moreover, EGFP was located at VSM cell borders in arterioles of transgenic mice carrying an EGFP-tagged Cx45. Expectedly, arteriolar conduction of dilations evoked by the endothelium-dependent agonist acetylcholine were not different between Cx45fl/fl:Nestin-Cre mice and controls carrying homozygously a floxed Cx45 gene (Cx45fl/fl). Surprisingly, the amplitude of locally initiated endothelium-independent constrictions (K+) and dilations (adenosine) declined similarly with distance in both genotypes indicating an intact VSM conduction pathway also in mice being deficient for Cx45 in VSM. Arterial pressure was not different between freely moving Cx45fl/fl and Cx45fl/fl:Nestin-Cre mice during day or night. We conclude that Cx45 is physiologically expressed in VSM, but not in EC in murine arterioles. However, Cx45 is dispensable for the conduction of vasomotor responses along these arterioles. Possibly, other Cx functionally replace the lack of Cx45 in VSM. The reported role of Cx45 in renin secretion does not seem to alter arterial pressure in freely moving mice.

C-terminal tagging with eGFP yields new insights into expression of connexin45 but prevents rescue of embryonic lethal connexin45-deficient mice

Connexin45 (Cx45) is a member of the connexin family which can form gap junction channels and is known to be expressed in several cell types in the embryonic as well as adult mouse including working cardiomyocytes and certain types of neurons. Until now its subcellular localization could not be unequivocally determined in certain tissues due to the lack of sensitive and specific antibodies. In order to investigate the localization of Cx45, we have generated a transgenic mouse expressing a fusion protein composed of Cx45 and eGFP under control of the endogenous Cx45 promoter using a bacterial artificial chromosome (BAC). In previous studies it had been shown that a C-terminal tag of connexin proteins only slightly altered the properties of gap junction channels in cultured cells and allowed direct visualization of the fusion protein. In the adult brain the expression of the Cx45eGFP protein was found in the subventricular zone in transient amplifying cells as well as in neuroblasts and ependymal cells. In addition Cx45eGFP is expressed in the atrial and ventricular working myocardium, i.e. regions of the heart where divergent results regarding Cx45 expression had previously been published. In the lung we identified Cx45eGFP in the smooth muscle cell layer of bronchioles. The Cx45eGFP transgene could not rescue embryonic lethality of Cx45-deficient mice, i.e. Cx45eGFP//Cx45(-/-) mice die around ED10.5 presumably due to altered properties of gap junction channels as a result of C-terminal tagging of Cx45.

Spatiotemporal Expression of Connexin 39 and −43 During Myoblast Differentiation in Cultured Cells and in the Mouse Embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C2C12 mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C2C12 cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.

Connexin39 deficient mice display accelerated myogenesis and regeneration of skeletal muscle

During muscle development and regeneration of skeletal muscle in mice connexin43 (Cx43) and connexin39 (Cx39) are specifically expressed: Cx43 in satellite cells and myoblasts, whereas Cx39 is exclusively expressed in myogenin-positive cells. We generated Cx39 deficient mice by replacing the coding region of the Gjd4 gene by DNA coding for the enhanced green fluorescent protein eGFP. Adult Cx39 deficient mice exhibit no obvious phenotypic alterations of skeletal muscle compared to wild type mice in the resting state. However, myogenesis in Cx39 deficient embryos is accelerated as indicated by increased myogenin expression on ED13.5 and ED16.5 and increased expression of Cx43 in developing skeletal muscle. In addition, the regeneration process of skeletal muscle in Cx39 deficient mice is accelerated as shown by a 2day earlier onset of MyoD and myogenin expression, relative to wild type littermates. Interestingly, Cx43 expression was also upregulated in Cx39 deficient mice during regeneration of skeletal muscle. We hypothesize that Cx43 may compensate for the loss of Cx39 during myogenesis and regeneration.

Essential Role of Gap Junctions During Development and Regeneration of Skeletal Muscle

1 Connexin and Pannexin Genes in the Mouse and Human Genome 1 Klaus Willecke, Jürgen Eiberger, Julia von Maltzahn 1.