Radoslaw Dobrowolski

Connexin-Caused Genetic Diseases and Corresponding Mouse Models

The human and mouse genomes contain 21 and 20 connexin genes, respectively. During the last 10-year period, genetic research on connexins has been stimulated by two parallel approaches: first, the characterization of genetic diseases that are caused by connexin mutations and, second, the generation and characterization of connexin knockout (null) mutated mice in which the coding region of nearly all connexin genes has been deleted. We summarize the current results of each of these two approaches. More recently, first results have been published in which connexin point mutations in human connexin genes were inserted at the corresponding position of the orthologous mouse gene. Under these conditions, the mutated connexin protein is expressed, in contrast to a connexin null mutation, and its interaction with other connexin isoforms or other connexin-binding proteins can be maintained. In this review, we discuss advantages and problems of such an approach and possible implications regarding the mechanism of the disease. The long-term goal is to understand the biologic function of each connexin isoform and the contribution of these proteins to the physiology of the corresponding organs in health and disease.

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels.

Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells

In the mouse genome, 20 connexin genes have been detected that code for proteins of high sequence identity in the two extracellular loops, especially six conserved cysteine residues. The mouse connexin23 (Cx23) gene (Gje1) differs from all other connexin genes in vertebrates, since it codes for a protein that contains only 4 instead of 6 cysteine residues in the extracellular loops. Recently, two zebrafish connexin genes (Cx23a and Cx23b) have been identified, and a mouse mutant in the Gje1 gene has been described that exhibits a developmental defect in the lens. Here, we have compared the Cx23 gene in different mammalian species and found no transcripts in cDNA libraries of primates. Furthermore, all primate genomes analyzed contain stop codons in the Cx23 sequence, indicating inactivation of the orthologous primate GJE1 gene. No Cx23 mRNA was found in human eye. In order to analyze the properties of mouse Cx23 channels, we isolated HeLa cell clones stably expressing wild-type mCx23 or mCx23 fused to eGFP. Cells expressing Cx23-eGFP demonstrated its insertion in the plasma membrane but no punctate staining in contacting membranes characteristic for junctional plaques. In addition, we tested whether Cx23 forms functional gap junction channels electrophysiologically in cell pairs as well as by microinjection of neurobiotin and found that mouse Cx23 did not form gap junction channels in HeLa cells. However, there was a significant release of ATP from different Cx23 HeLa cell clones, even in the presence of normal culture medium with high calcium ion concentration, suggesting a hemichannel-based function of Cx23. Therefore, Cx23 seems to share functional properties with pannexin (hemi) channels rather than gap junction channels of other connexins.

The Connexin40A96S mutation from a patient with atrial fibrillation causes decreased atrial conduction velocities and sustained episodes of induced atrial fibrillation in mice

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient.

Human connexin31.9, unlike its orthologous protein connexin30.2 in the mouse, is not detectable in the human cardiac conduction system

In the human heart connexin(Cx)40, Cx43 and Cx45-containing gap junctional channels electrically couple cardiomyocytes, forming a functional syncytium. In the mouse heart, additionally, Cx30.2-containing gap junctions have been detected in the atrioventricular node where they are implicated, together with Cx45, in impulse delay. However, whether the human ortholog of Cx30.2, Cx31.9, is expressed in the human heart has not previously been investigated. We therefore generated Cx31.9 specific antibodies to test for the expression of Cx31.9 in the human heart. These antibodies recognized the Cx31.9 protein in HeLaCx31.9 transfectants by immunofluorescence and immunoblot analyses. However, we did not find punctate Cx31.9 specific immunofluorescence signals in the working myocardium or in the impulse generation and conduction system of adult or fetal human heart. Complementary immunoblot analyses did not reveal Cx31.9 protein in the adult atrial or ventricular myocardium. We conclude that the Cx31.9 protein, unlike its counterpart in the mouse, is not expressed in detectable quantities and is thus unlikely to contribute to the impulse generation and conduction system or the working myocardium of the human heart.

The TSG101 protein binds to connexins and is involved in connexin degradation

Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

Lack of Laminin γ1 in Embryonic Stem Cell‐Derived Cardiomyocytes Causes Inhomogeneous Electrical Spreading Despite Intact Differentiation and Function

Laminins form a large family of extracellular matrix (ECM) proteins, and their expression is a prerequisite for normal embryonic development. Herein we investigated the role of the laminin γ1 chain for cardiac muscle differentiation and function using cardiomyocytes derived from embryonic stem cells deficient in the LAMC1 gene. Laminin γ1 (−/−) cardiomyocytes lacked basement membranes (BM), whereas their sarcomeric organization was unaffected. Accordingly, electrical activity and hormonal regulation were found to be intact. However, the inadequate BM formation led to an increase of ECM deposits between adjacent cardiomyocytes, and this resulted in defects of the electrical signal propagation. Furthermore, we also found an increase in the number of pacemaker areas. Thus, although laminin and intact BM are not essential for cardiomyocyte development and differentiation per se, they are required for the normal deposition of matrix molecules and critical for intact electrical signal propagation. STEM CELLS 2009;27:88–99

Neuronal connexin-36 can functionally replace connexin-45 in mouse retina but not in the developing heart

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is co-expressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36.

Hepatoma‐derived growth factor (HDGF) is dispensable for normal mouse development

Hepatoma‐derived growth factor (HDGF) is suggested to be involved in organ development and exhibits proliferative, angiogenic, and neurotrophic activity. The in vivo functions are, however, so far unknown. In this study, we generated HDGF‐deficient mice, in which parts of the HDGF gene were replaced by a gene encoding green fluorescent protein (eGFP). HDGF−/− mice are viable with no apparent morphological abnormalities. Cultured HDGF‐deficient dermal fibroblasts show unaltered proliferation rates and cell‐cycle distributions. In contrast to previous studies, our data demonstrate that signal pathways involved in the response to extracellular HDGF do not depend on the presence of intracellular HDGF. Contrary to the reported role of HDGF as a modulator of apoptosis, similar apoptotic rates were found between wild‐type and HDGF‐deficient fibroblasts following tumor necrosis factor α (TNFα) ‐induced apoptosis or cellular stress. The lack of obvious biochemical and morphological phenotypes in HDGF‐deficient mice demonstrates that in vivo HDGF is dispensable for normal development in mice. Developmental Dynamics 237:1875–1885, 2008.