RESUMEN
RATIONALE: Delivery of Cx43 (connexin 43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking and that Cx43 has a 20 kDa internally translated small C terminus isoform, GJA1-20k (Gap Junction Protein Alpha 1- 20 kDa), which is required for full-length Cx43 trafficking, but by an unknown mechanism. OBJECTIVE: We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. METHODS AND RESULTS: Using an in vivo Adeno-associated virus serotype 9-mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin without affecting actin protein expression and that GJA1-20k complexes with both actin and tubulin. We also found that filamentous actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from latrunculin A disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo Adeno-associated virus serotype 9-mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. CONCLUSIONS: The internally translated GJA1-20k isoform stabilizes actin filaments, which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell-cell coupling in instances of anticipated myocardial ischemia.
Asunto(s)
Actinas/metabolismo , Conexina 43/metabolismo , Técnicas de Transferencia de Gen , Miocitos Cardíacos/metabolismo , Actinas/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Conexina 43/genética , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/genética , Microtúbulos/metabolismo , Técnicas de Cultivo de Órganos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha18H/a18H). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67+ proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha18H/a18H animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Homeostasis/fisiología , Intestino Delgado/metabolismo , Ubiquitina/metabolismo , Animales , Mucosa Intestinal/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through ß-adrenergic receptor (ß-AR)-mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates CaV1.2 delivery, we explored whether ß-AR-regulated RyRs are also affected by BIN1. METHODS AND RESULTS: Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the ß-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. Acute ß-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1. CONCLUSIONS: On ß-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Señalización del Calcio/fisiología , Isoproterenol/farmacología , Proteínas del Tejido Nervioso/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Agonistas Adrenérgicos beta/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a ß-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.
Asunto(s)
Arritmias Cardíacas/genética , Conexina 43/genética , Hipertrofia Ventricular Izquierda/genética , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Actinas/genética , Actinas/metabolismo , Animales , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Femenino , Uniones Comunicantes/metabolismo , Uniones Comunicantes/patología , Regulación de la Expresión Génica , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Masculino , Ratones , Ratones Transgénicos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/patología , Fenotipo , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Connexin 43 (Cx43), a product of the GJA1 gene, is a gap junction protein facilitating intercellular communication between cardiomyocytes. Cx43 protects the heart from ischemic injury by mechanisms that are not well understood. GJA1 mRNA can undergo alternative translation, generating smaller isoforms in the heart, with GJA1-20k being the most abundant. Here, we report that ischemic and ischemia/reperfusion (I/R) injuries upregulate endogenous GJA1-20k protein in the heart, which targets to cardiac mitochondria and associates with the outer mitochondrial membrane. Exploring the functional consequence of increased GJA1-20k, we found that AAV9-mediated gene transfer of GJA1-20k in mouse hearts increases mitochondrial biogenesis while reducing mitochondrial membrane potential, respiration, and ROS production. By doing so, GJA1-20k promotes a protective mitochondrial phenotype, as seen with ischemic preconditioning (IPC), which also increases endogenous GJA1-20k in heart lysates and mitochondrial fractions. As a result, AAV9-GJA1-20k pretreatment reduces myocardial infarct size in mouse hearts subjected to in vivo ischemic injury or ex vivo I/R injury, similar to an IPC-induced cardioprotective effect. In conclusion, GJA1-20k is an endogenous stress response protein that induces mitochondrial biogenesis and metabolic hibernation, preconditioning the heart against I/R insults. Introduction of exogenous GJA1-20k is a putative therapeutic strategy for patients undergoing anticipated ischemic injury.
Asunto(s)
Conexina 43/metabolismo , Mitocondrias/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/metabolismo , Daño por Reperfusión/complicaciones , Animales , Conexina 43/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/genética , Células HEK293 , Humanos , Preparación de Corazón Aislado , Masculino , Ratones , Mitocondrias/patología , Infarto del Miocardio/etiología , Infarto del Miocardio/prevención & control , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Biogénesis de Organelos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/terapia , Resultado del TratamientoRESUMEN
Connexin 43 (Cx43, encoded by GJA1) is a cell-cell communication gap junction protein expressed in all organ systems. It was recently found that GJA1 mRNA undergoes alternative translation to generate N-terminal truncated isoforms, of which GJA1-20k is the most abundant. Here we report a surprising finding that, unlike full length GJA1-43k, GJA1-20k has a strong tropism for mitochondria. Exploring function, we found that GJA1-20k appears to be an organelle chaperone and that overexpression of GJA1-20k is sufficient to rescue mitochondrial localization to the cell periphery upon exposure to hydrogen peroxide, which effectively limits the network fragmentation that occurs with oxidative stress. By high-resolution fluorescent imaging and electron microscopy, we determined that GJA1-20k is enriched at the interface between mitochondria and microtubules, appearing to load organelles for transport. Mutagenesis experiments revealed that although the microtubule-binding domain (MTBD) in GJA1-20k is not necessary for protein localization to mitochondria, the MTBD is essential for GJA1-20k to facilitate mitochondrial transport and maintain mitochondrial localization at the periphery. These results reveal an unexpected role for the alternatively translated isoform of the Cx43 gap junction protein, GJA1-20k, which is to facilitate microtubule-based mitochondrial transport and to maintain mitochondrial network integrity during cellular stress.