Internal translation of Gja1 (Connexin43) to produce GJA1-20k: Implications for arrhythmia and ischemic-preconditioning.

Internal translation is a form of post-translation modification as it produces different proteins from one mRNA molecule by beginning translation at a methionine coding triplet downstream of the first methionine. Internal translation can eliminate domains of proteins that otherwise restrict movement or activity, thereby creating profound functional diversity. Connexin43 (Cx43), encoded by the gene Gja1, is the main gap junction protein necessary for propagating action potentials between adjacent cardiomyocytes. Gja1 can be internally translated to produce a peptide 20 kD in length named GJA1-20k. This review focuses on the role of GJA1-20k in maintaining cardiac electrical rhythm as well as in ischemic preconditioning (IPC). Connexin43 is the only ion channel we are aware that has been reported to be subject to internal translation. We expect many other ion channels also undergo internal translation. The exploration of post-translational modification of ion channels, and in particular of internal translation, has the potential to greatly increase our understanding of both canonical and non-canonical ion channel biology.

Connexin 43 in Mitochondria: What Do We Really Know About Its Function?

Connexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.

GJA1-20k and Mitochondrial Dynamics.

Connexin 43 (Cx43) is the primary gap junction protein of mammalian heart ventricles and is encoded by the gene Gja1 which has a single coding exon and therefore cannot be spliced. We previously identified that Gja1 mRNA undergoes endogenous internal translation initiated at one of several internal AUG (M) start codons, generating N-terminal truncated protein isoforms that retain the C-terminus distal to the start site. GJA1-20k, whose translation initiates at mRNA M213, is usually the most abundant isoform in cells and greatly increases after ischemic and metabolic stress. GJA1-20k consists of a small segment of the last transmembrane domain and the complete C-terminus tail of Cx43, with a total size of about 20 kDa. The original role identified for GJA1-20k is as an essential subunit that facilitates the trafficking of full-length Cx43 hexameric hemichannels to cell-cell contacts, generating traditional gap junctions between adjacent cells facilitating, in cardiac muscle, efficient spread of electrical excitation. GJA1-20k deficient mice (generated by a M213L substitution in Gja1) suffer poor electrical coupling between cardiomycytes and arrhythmogenic sudden death two to 4 weeks after their birth. We recently identified that exogenous GJA1-20k expression also mimics the effect of ischemic preconditioning in mouse heart. Furthermore, GJA1-20k localizes to the mitochondrial outer membrane and induces a protective and DRP1 independent form of mitochondrial fission, preserving ATP production and generating less reactive oxygen species (ROS) under metabolic stress, providing powerful protection of myocardium to ischemic insult. In this manuscript, we focus on the detailed roles of GJA1-20k in mitochondria, and its interaction with the actin cytoskeleton.

GJA1-20K Enhances Mitochondria Transfer from Astrocytes to Neurons via Cx43-TnTs After Traumatic Brain Injury.

Astrocytes are crucial in neural protection after traumatic brain injury (TBI), a global health problem causing severe brain tissue damage. Astrocytic connexin 43 (Cx43), encoded by GJA1 gene, has been demonstrated to facilitate the protection of astrocytes to neural damage with unclear mechanisms. This study aims to explore the role of GJA1-20K/Cx43 axis in the astrocyte-neuron interaction after TBI and the underlying mechanisms. Primarily cultured cortical neurons isolated from embryonic C57BL/6 mice were treated by compressed nitrogen-oxygen mixed gas to simulate TBI-like damage in vitro. The transwell astrocyte-neuron co-culture system were constructed to recapitulate the interaction between the two cell types. Quantitative PCR was applied to analyze mRNA level of target genes. Western blot and immunofluorescence were conducted to detect target proteins expression. GJA1-20K overexpression significantly down-regulated the expression of phosphorylated Cx43 (p-Cx43) without affecting the total Cx43 protein level. Besides, GJA1-20K overexpression obviously enhanced the dendrite length, as well as the expression levels of function and synthesis-related factors of mitochondria in damaged neurons. GJA1-20K up-regulated functional Cx43 expression in astrocytes, which promoted mitochondria transmission from astrocytes to neurons which might be responsible to the protection of astrocyte to neurons after TBI-like damage in vitro.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

GJA1-20k attenuates Ang II-induced pathological cardiac hypertrophy by regulating gap junction formation and mitochondrial function.

Cardiac hypertrophy (CH) is characterized by an increase in cardiomyocyte size, and is the most common cause of cardiac-related sudden death. A decrease in gap junction (GJ) coupling and mitochondrial dysfunction are important features of CH, but the mechanisms of decreased coupling and energy impairment are poorly understood. It has been reported that GJA1-20k has a strong tropism for mitochondria and is required for the trafficking of connexin 43 (Cx43) to cell-cell borders. In this study, we investigated the effects of GJA1-20k on Cx43 GJ coupling and mitochondrial function in the pathogenesis of CH. We performed hematoxylin-eosin (HE) and Masson staining, and observed significant CH in 18-week-old male spontaneously hypertensive rats (SHRs) compared to age-matched normotensive Wistar-Kyoto (WKY) rats. In cardiomyocytes from SHRs, the levels of Cx43 at the intercalated disc (ID) and the expression of GJA1-20k were significantly reduced, whereas JAK-STAT signaling was activated. Furthermore, the SHR rats displayed suppressed mitochondrial GJA1-20k and mitochondrial biogenesis. Administration of valsartan (10 mg· [Formula: see text] d-1, i.g., for 8 weeks) prevented all of these changes. In neonatal rat cardiomyocytes (NRCMs), overexpression of GJA1-20k attenuated Ang II-induced cardiomyocyte hypertrophy and caused elevated levels of GJ coupling at the cell-cell borders. Pretreatment of NRCMs with the Jak2 inhibitor AG490 (10 µM) blocked Ang II-induced reduction in GJA1-20k expression and Cx43 gap junction formation; knockdown of Jak2 in NRCMs significantly lessened Ang II-induced cardiomyocyte hypertrophy and normalized GJA1-20k expression and Cx43 gap junction formation. Overexpression of GJA1-20k improved mitochondrial membrane potential and respiration and lowered ROS production in Ang II-induced cardiomyocyte hypertrophy. These results demonstrate the importance of GJA1-20k in regulating gap junction formation and mitochondrial function in Ang II-induced cardiomyocyte hypertrophy, thus providing a novel therapeutic strategy for patients with cardiomyocyte hypertrophy.

Overexpression of Astrocytes-Specific GJA1-20k Enhances the Viability and Recovery of the Neurons in a Rat Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a devastating actuality in clinics worldwide. It is estimated that approximately 10 million people among the world suffer from TBI each year, and a considerable number of patients will be temporarily or permanently disabled or even die due to this disease. Astrocytes play a very important role in the repair of brain tissue after TBI, including the formation of a neuroprotective barrier, inhibition of brain edema, and inhibition of normal nerve cell apoptosis. However, the detailed mechanism underlying this protective effect is still unclear. To investigate the regulatory factors of astrocytes to other neurons post-TBI, we established a TBI rat model and used the AAV to mediate the overexpression of GJA1-20k in astrocytes of rats. And functionally, the specific overexpression of GJA1-20k in astrocytes promoted the viability and recovery of neurons in TBI. Mechanistically, the astrocytes-specific upregulation of GJA1-20k protected the function of mitochondria in neurons of FPI rats, thus suppressing the apoptosis of the damaged neurons. We hereby reported that astrocytes-specific overexpression of GJA1-20k enhanced the viability and recovery of the neurons in TBI through regulating their mitochondrial function.

Astrocytes-derived exosomes induce neuronal recovery after traumatic brain injury via delivering gap junction alpha 1-20 k.

Astrocytes are more resistant to ischemia and hypoxia in the acute phase of brain injury after traumatic brain injury (TBI). Previous study showed that gap junction alpha 1 (GJA1) phosphorylation can increase the survival of damaged astrocytes. The GJA1-20 k expression in neurons co-culture with astrocytes was positively correlated with exosomes uptake. This study aims to explore the effect of exogenous GJA1-20 k carried by astrocyte-derived exosomes on neurons apoptosis and mitochondrial function after TBI. Astrocytes were co-cultured with the neuron with/without damage from air pressure. Exosomes were isolated, extracted from the culture medium by differential ultra-centrifugation, and verified by electron microscopy. Immunofluorescence staining, tunnel, western blot were employed to detect exosomes marker CD60, apoptosis, and mitochondrial function related protein expression and GJA1-20 k in cell culture. A rat model of hydraulic injury TBI was built, and exosomes was transferred. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry staining of Nissl and microtubule associated protein 2 were used to detect the brain damage. A transwell stereo culture model of astrocytes and TBI-like injured neuron was constructed. The exosomes derived from astrocytes promoted the recovery of damaged neuron by in vitro exosome treatment. Compared with GJA1-20 k knockout exosome control group, GJA1-20 k exosomes were uptaken by neuron and downregulated the apoptosis rate and upregulated mitochondrial function to promote neuronal recovery. Finally, the results were validated by TTC staining and damaged tissue sections of rat TBI model. This study contributes to a better understanding of the astrocyte-neuron protection mechanism in TBI and provides a potential new target for the treatment of TBI.