Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

HSPB7 is belonged to small heat-shock protein (HSPB) family and considered to function as a co-chaperone, which prevents protein aggregation and maintains protein structure. Single-nucleotide polymorphisms of HSPB7 associated with sporadic cardiomyopathy and heart failure have been identified in human patients. Additionally, HSPB7 is constitutively expressed in heart and rapidly increased in blood plasma after myocardial infarction, suggesting a functional role in the heart. In this study, we found that HSPB7 is highly colocalized with N-cadherin during the assembly and maturation of intercalated disc, suggesting that HSPB7 may involve in organizing and maintaining the cardiac cytoarchitecture. To elucidate the physiological function of HSPB7 in the adult heart, we generated a cardiac-specific inducible HSPB7 knockout mouse. Ablation of HSPB7 in the cardiomyocyte rapidly leads to heart failure, abnormal conduction properties and sudden arrhythmias death. Loss of HSPB7 did not cause significant changes in the organization of contractile proteins in sarcomeres, whereas severe abnormality in the intercalated disc was detected. The expression of connexin 43, a gap-junction protein located at the intercalated disc, was downregulated in HSPB7 knockout cardiomyocytes. Mislocalizations of desmoplakin (desmosomal proteins), and N-cadherin (adherens junction proteins) were also observed in the HSPB7 CKO hearts. Furthermore, filamin C, the interaction protein of HSPB7, was mislocalized and aggregated in HSPB7 mutant cardiomyocytes. The expressivity of the phenotype in the HSPB7 CKO mice is similar to human arrhythmogenic cardiomyopathy patients. Conclusively, we provide the first study characterizing HSPB7 as an intercalated disc protein. Our findings demonstrate that HSPB7 plays an essential role to maintain the structure and function of gap-junction complexes and intercalated disc and has vital implications for human heart disease.

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Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart

Journal of Cell Science ◽

10.1242/jcs.115.8.1623 ◽

2002 ◽

Vol 115 (8) ◽

pp. 1623-1634 ◽

Cited By ~ 2

Author(s):

M. Celeste Ferreira-Cornwell ◽

Yang Luo ◽

Navneet Narula ◽

Jennifer M. Lenox ◽

Melanie Lieberman ◽

...

Keyword(s):

Cell Adhesion ◽

Transgenic Mice ◽

Dilated Cardiomyopathy ◽

Cyclin D1 ◽

Connexin 43 ◽

Ectopic Expression ◽

Adherens Junction ◽

Transgenic Animals ◽

Intercalated Disc ◽

E Cadherin

The contractile force of the cardiomyocyte is transmitted through the adherens junction, a component of the intercalated disc, enabling the myocardium to function as a syncytium. The cadherin family of cell adhesion receptors, located in the adherens junction, interact homophilically to mediate strong cell-cell adhesion. Ectopic expression of cadherins is associated with changes in tumor cell behavior and pathology. To examine the effect of cadherin specificity on cardiac structure and function, we expressed either the epithelial cadherin, E-cadherin, or N-cadherin in the heart of transgenic mice. E-cadherin was localized to the intercalated disc structure in these animals similar to endogenous N-cadherin. Both N- and E-cadherin transgenic animals developed dilated cardiomyopathy. However, misexpression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice. A dramatic decrease in connexin 43 was associated with the hypertrophic response in E-cadherin transgenic mice. Myofibril organization appeared normal although, vinculin, which normally localizes to the intercalated disc, was redistributed to the cytoplasm in the E-cadherin transgenic mice. Furthermore, E-cadherin induced cyclin D1, nuclear reduplication, and karyokinesis in the absence of cytokinesis, resulting in myocytes with two closely opposed nuclei. By contrast, N-cadherin overexpressing transgenic mice did not exhibit an increase in cyclin D1,suggesting that E-cadherin may provide a specific growth signal to the myocyte. This study demonstrates that modulation of cadherin-mediated adhesion can lead to dilated cardiomyopathy and that E-cadherin can stimulate DNA replication in myocytes normally withdrawn from the cell cycle.

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Abstract P361: Reduced Cardiac Tmem65 In Mouse Hearts Results In Intercalated Disc Defects And Eventual Dilated Cardiomyopathy With Cardiac Fibrosis

Circulation Research ◽

10.1161/res.129.suppl_1.p361 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Allen C Teng ◽

Liyang Gu ◽

Michelle Di Paola ◽

Zachary Williams ◽

Aaron Au ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Sodium Channel ◽

Connexin 43 ◽

Cardiac Fibrosis ◽

Quantitative Polymerase Chain Reaction ◽

Physical Interaction ◽

Intercalated Disc ◽

Voltage Gated Sodium Channel ◽

Voltage Gated

The intercalated disc (ICD) is unique membrane structure that is indispensable to normal heart function. However, its structural organization is not well understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin 43 (Cx43) localization in cultured mouse neonatal cardiomyocytes. Here, we investigated the role of Tmem65 in ICD organization in vivo . A mouse model was established by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 (or scrambled) shRNA. Quantitative polymerase chain reaction (qPCR) and immunoblots confirmed greater than 85% reduction in Tmem65 expression (7.1±0.7% remained for Tmem65 proteins; 14.4±2.5% remained for Tmem65 transcripts, n =4) in mouse ventricles compared to control hearts. Tmem65 knockdown (KD) mice exhibited heart failure-like symptoms as early as 3 weeks post viral administration. Specifically, Tmem65 KD mice developed eccentric hypertrophic cardiomyopathy in 3 weeks and dilated cardiomyopathy with severe cardiac fibrosis in 7 weeks, as confirmed by H&E and Masson’s Trichrome staining. Echocardiography and electrocardiography, respectively, showed depressed hemodynamics (19.27±1.46ml/min for cardiac output in control hearts vs. 6.63±0.52ml/min for Tmem65 KD hearts, n =6) and impaired conduction, including prolonged PR (22.7±1.85ms in control hearts vs. 28.89±3.85ms in Tmem65 KD hearts, n≥8), QRS intervals (10.47±0.42ms in control hearts vs. 16.35±0.36ms in Tmem65 KD hearts, n≥8), and slowed heart rate (415±10bpm in control hearts vs. 347±16bpm in Tmem65 KD hearts, n≥8) in Tmem65 KD mouse hearts. Immunoprecipitation and super-resolution microscopy confirmed the physical interaction and localization between Tmem65 and voltage-gated sodium channel β subunit (β1) at the ICD and this interaction was evidently required for the establishment of perinexal nanodomains and voltage-gated sodium channel 1.5 (NaV1.5) localization to the ICD. Disrupting Tmem65 function, thus, impaired perinexal structure, reduced conduction velocity, and ultimately resulted in cardiomyopathy in vivo .

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Gap junctions–guards of excitability

Biochemical Society Transactions ◽

10.1042/bst20150059 ◽

2015 ◽

Vol 43 (3) ◽

pp. 508-512 ◽

Cited By ~ 16

Author(s):

Line Waring Stroemlund ◽

Christa Funch Jensen ◽

Klaus Qvortrup ◽

Mario Delmar ◽

Morten Schak Nielsen

Keyword(s):

Sodium Channel ◽

Crucial Role ◽

Connexin 43 ◽

Current Evidence ◽

Intercalated Disc ◽

Intercalated Discs ◽

Cardiac Sodium Channel ◽

Junctional Protein ◽

Gap Junctional

Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane.

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Remodelling of myocardial intercalated disc protein connexin 43 causes increased susceptibility to malignant arrhythmias in ARVC/D patients

Forensic Science International ◽

10.1016/j.forsciint.2017.02.020 ◽

2017 ◽

Vol 275 ◽

pp. 14-22 ◽

Cited By ~ 6

Author(s):

Xiao Chen ◽

Liang Chen ◽

Zhenglian Chen ◽

Xinshan Chen ◽

Jiangping Song

Keyword(s):

Connexin 43 ◽

Intercalated Disc ◽

Malignant Arrhythmias ◽

Increased Susceptibility

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Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice

Circulation ◽

10.1161/circulationaha.120.050218 ◽

2020 ◽

Vol 142 (22) ◽

pp. 2155-2171

Author(s):

Harald Herrmann ◽

Eva Cabet ◽

Nicolas R. Chevalier ◽

Julia Moosmann ◽

Dorothea Schultheis ◽

...

Keyword(s):

Connexin 43 ◽

Molecular Mechanisms ◽

Cell Transfection ◽

Intercalated Disc ◽

Dual Functional ◽

Intercalated Discs ◽

Cardiac Phenotype ◽

Functional States ◽

Explanted Heart

Background: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. Methods: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. Results: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. Conclusions: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

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Abstract 18420: Myozap-deficiency Inhibits SRF Signaling and Causes Severe Cardiomyopathy in Response to Pathological Biomechanical Stress in vivo

Circulation ◽

10.1161/circ.130.suppl_2.18420 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Ashraf Y Rangrez ◽

Derk Frank ◽

Reza Poyanmehr ◽

Alexander Bernt ◽

Matthias Eden ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Hypertrophy ◽

Connexin 43 ◽

Target Genes ◽

Clinical Signs ◽

Structural Defects ◽

Intercalated Disc ◽

Biomechanical Stress ◽

Cardiac Phenotype

Background: We recently showed that the intercalated disc (ID) protein Myozap activates Rho-dependent SRF signaling both in vitro, and in vivo. Conversely, knockdown of its ortholog in Zebrafish led to severe contractile dysfunction and cardiomyopathy. Methods and Results: We generated a Myozap null mutant (MZP-ko) by global deletion of Myozap in mice. The absence of Myozap caused neither structural defects nor a baseline cardiac phenotype and led only to mild cardiac hypertrophy upon aging. Of note, induction of SRF target genes and the activation of SRF per se were markedly inhibited in MZP-ko mice. Nevertheless, biomechanical stress induced by transverse aortic constriction (TAC) triggered an excessive increase in cardiac hypertrophy (43% or 36% increased heart wt:body wt or LV wt:body wt ratios, p<0.001), an increased cell surface area, as well as accelerated fibrosis, followed by “super”- induction of the hypertrophic gene program (ANF/BNP). Moreover, MZP-ko mice revealed a severe reduction of fractional shortening (average %FS for MZP-ko 14.5% compared to 33.5% for wild-type littermates, p<0.001) and clinical signs of heart failure (54% increase in lung/body weights, p<0.001) which also caused a profound increase in mortality in response to TAC. Furthermore, expression of other ID proteins like N-Cadherin, Desmoplakin and Connexin 43 was found significantly altered upon pressure overload in MZP-ko mice. Finally, we observed a downregulation of Dysbindin, a novel interaction partner of Myozap and known inducer of ERK1/2 signaling in TAC operated MZP-ko mice. Consistently, activation of ERK1/2 was blunted in MZP-ko mice after TAC. Conclusions: We here show that myozap deficiency in vivo inhibits SRF- and ERK1/2-signaling leading to a maladaptative response to increased biomechanical stress, followed by cardiomyopathy, heart failure and cardiac death. Moreover, myozap deficiency severely altered the expression of its direct ID interaction partners such as Dysbindin and Desmoplakin. In a broader perspective, our data identify signaling at the level of the intercalated disc as a critical component of cardiac remodeling.

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