Connexin43 and the regulation of intercalated disc function.
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ABSTRACT:
SUBMITTER: Delmar M
PROVIDER: S-EPMC3289726 | biostudies-literature | 2012 May
REPOSITORIES: biostudies-literature
Publications
Connexin43 and the regulation of intercalated disc function.
Delmar Mario M Liang Feng-Xia FX
Heart rhythm 20111102 5
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Project description:RationaleThe early description of the intercalated disc defined 3 structures, all of them involved in cell-cell communication: desmosomes, gap junctions, and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells also populate the intercalated disc. Key among them is the voltage-gated sodium channel complex. An important component of this complex is the cytoskeletal adaptor protein Ankyrin-G (AnkG).ObjectiveTo test the hypothesis that AnkG partners with desmosome and gap junction molecules and exerts a functional effect on intercellular communication in the heart.Methods and resultsWe used a combination of microscopy, immunochemistry, patch-clamp, and optical mapping to assess the interactions between AnkG, Plakophilin-2, and Connexin43. Coimmunoprecipitation studies from rat heart lysate demonstrated associations between the 3 molecules. With the use of siRNA technology, we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Connexin43 as well as a decrease in intercellular adhesion strength and electric coupling. Regulation of AnkG and of Na(v)1.5 by Plakophilin-2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed.ConclusionsThese experiments support the hypothesis that AnkG is a key functional component of the intercalated disc at the intersection of 3 complexes often considered independent: the voltage-gated sodium channel, gap junctions, and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy) are discussed.
| S-EPMC3139453 | biostudies-literature
Project description:Xin was first cloned using differential mRNA display from the developing chicken heart. Chick Xin (cXin) participates in a BMP-Nkx2.5-MEF2C pathway to regulating cardiac morphogenesis. Through subsequent EST database searches and cDNA cloning, two mouse Xin genes, mXinα and mXinβ were identified and cloned. The human homologue of mXinα (named Cmya1) was mapped to chromosome 3p21.2-p21.3 by radiation hybrid analysis and recently to 3p22.2 by DNA sequencing, which is near the loci for a dilated cardiomyopathy with conduction defect-2 and arrhythmogenic right ventricular dysplasia-5. The predicted human homologue of mXinβ (named Cmya3) was mapped to chromosome 2q24.3 by DNA sequencing. Predicted Xin proteins all contain a novel 16-amino acid repeating unit (Xin repeat), a putative DNA binding domain and nuclear localization signal, as well as a proline-rich region. All three Xin genes from chick and mouse have a similar tissue expression profile, which is restricted to striated muscle. The expression of mXinα in Nkx2.5 or MEF2C knockout mouse embryos was drastically reduced, suggesting that mXinα is a downstream target of the Nkx2.5 and MEF2C transcription factors. On the other hand, the expression of mXin was up-regulated when mice were subjected to pressure overload-induced cardiac hypertrophy. Xin protein co-localizes with N-cadherin and β-catenin throughout mouse embryogenesis and into adulthood. Furthermore, mXinα appears to interact directly with β-catenin. The Xin repeats bind to actin filaments and may also organize microfilaments into networks. These results may suggest that Xin acts by integrating adhesion, by organizing actin filament arrangement at the insertion sites, and by regulating Wnt/β-catenin-and N-cadherin-mediated signaling pathways required for cardiac development and cardiac function.
| S-EPMC1458968 | biostudies-literature
Project description:BackgroundMany signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd(2+)), we tested this hypothesis.Principal findingsIncubation of Cx43-transfected LLC-PK1 cells with Cd(2+) led to an increased expression of Cx43. This effect of Cd(2+) was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd(2+) on JNK activation and Cx43 expression. Indeed, Cd(2+) induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd(2+).ConclusionsCollectively, our results thus indicate that Cd(2+)-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.
| S-EPMC3584027 | biostudies-literature
Project description:AimsIt is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant 'Cx43D378stop') led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein 'end-binding 1' (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa.Methods and resultsSuper-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules.ConclusionsNaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.
| S-EPMC4296112 | biostudies-literature
Project description:Alterations in myocardial structure and reduced cardiomyocyte adhesions have been previously described in dilated cardiomyopathy (DCM). We studied the transcriptome of cell adhesion molecules in these patients and their relationships with left ventricular (LV) function decay. We also visualized the intercalated disc (ID) structure and organization. The transcriptomic profile of 23 explanted LV samples was analyzed using RNA-sequencing (13 DCM, 10 control [CNT]), focusing on cell adhesion genes. Electron microscopy analysis to visualize ID structural differences and immunohistochemistry experiments of ID proteins was also performed. RT-qPCR and western blot experiments were carried out on ID components. We found 29 differentially expressed genes, most of all, constituents of the ID structure. We found that the expression of GJA3, DSP and CTNNA3 was directly associated with LV ejection fraction (r = 0.741, P = 0.004; r = 0.674, P = 0.011 and r = 0.565, P = 0.044, respectively), LV systolic (P = 0.003, P = 0.003, P = 0.028, respectively) and diastolic dimensions (P = 0.006, P = 0.001, P = 0.025, respectively). Electron microscopy micrographs showed a reduced ID convolution index and immunogold labeling of connexin 46 (GJA gene), desmoplakin (DSP gene) and catenin α-3 (CTNNA3 gene) proteins in DCM patients. Moreover, we observed that protein and mRNA levels analyzed by RT-qPCR of these ID components were diminished in DCM group. In conclusion, we report significant gene and protein expression changes and found that the ID components GJA3, DSP and CTNNA3 were highly related to LV function. Microscopic observations indicated that ID is structurally compromised in these patients. These findings give new data for understanding the ventricular depression that characterizes DCM, opening new therapeutic perspectives for these critically diseased patients.
| S-EPMC5608295 | biostudies-literature
Project description:RationaleZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined.ObjectiveWe aim to determine the role of ZO-1 in cardiac function.Methods and resultsInducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction.ConclusionsZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.
| S-EPMC7334106 | biostudies-literature
Project description:The development of the normal human heart, ranging from gestational age to the mature adult heart, relies on a very delicate and timely orchestrated order of processes. One of the most striking alterations in time is the gradual extinction of the ability for cardiomyocytes to proliferate. Once passing this event, cardiomyocytes grow and increase in contractile strength by means of physiological hypertrophy. This process, importantly, seems to depend on an adequate development of electromechanical coupling that is achieved by the appropriate formation of the intercellular junction named the intercalated disc (ICD). In this report, we describe two sudden death cases of young and apparently healthy-born individuals without external abnormalities compared to an age-matched control. Histological examination, including the comparison with the age-matched and histology-matched controls, showed a disturbed formation of the protein machinery composing the electromechanical junctions at the ICD and an increased nuclei count for both patients. As a cause or consequence, cardiomyocytes in both sudden death cases showed signs of a delayed developmental stage, presumably resulting in an exaggerated degree of hyperplasia.
| S-EPMC10455643 | biostudies-literature
Project description:Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc, resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.
| S-EPMC8011902 | biostudies-literature
Project description:Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and 'angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.
| S-EPMC4735805 | biostudies-literature
Project description:HSPB7 is a member of the small heat-shock protein (HSPB) family and is expressed in the cardiomyocytes from cardiogenesis onwards. A dramatic increase in HSPB7 is detected in the heart and blood plasma immediately after myocardial infarction. Additionally, several single-nucleotide polymorphisms of HSPB7 have been identified to be associated with heart failure caused by cardiomyopathy in human patients. Although a recent study has shown that HSPB7 is required for maintaining myofiber structure in skeletal muscle, its molecular and physiological functions in the heart remain unclear. In the present study, we generated a cardiac-specific inducible HSPB7 knockout mouse and demonstrated that the loss of HSPB7 in cardiomyocytes results in rapid heart failure and sudden death. The electrocardiogram showed cardiac arrhythmia with abnormal conduction in the HSPB7 mutant mice before death. In HSPB7 CKO cardiomyocytes, no significant defect was detected in the organization of contractile proteins in sarcomeres, but a severe structural disruption was observed in the intercalated discs. The expression of connexin 43, a gap-junction protein located at the intercalated discs, was downregulated in HSPB7 knockout cardiomyocytes. Mislocalization of desmoplakin, and N-cadherin, the intercalated disc proteins, was also observed in the HSPB7 CKO hearts. Furthermore, filamin C, the interaction protein of HSPB7, was upregulated and aggregated in HSPB7 mutant cardiomyocytes. In conclusion, our findings characterize HSPB7 as an intercalated disc protein and suggest it has an essential role in maintaining intercalated disc integrity and conduction function in the adult heart.
| S-EPMC5587339 | biostudies-literature