GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs

Abstract

Rationale Delivery of connexin 43 (Cx43) 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 20kDa internally translated small C-terminus isoform (GJA1-20k) 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 AAV9-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 (F-actin) without affecting actin protein expression, and that GJA1-20k complexes with both actin and tubulin. We also found that F-actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A (LatA) disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from LatA disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo AAV9-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.

Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury

“…While the existence of GJA1-20k can help explain the role of Cx43 in protection of cardiac metabolism (30), future studies are needed to understand the tropism of GJA1-20k to mitochondria and how it induces biogenesis and reduces respiration simultaneously. In other studies, it has been identified that GJA1-20k is necessary for the trafficking of full-length Cx43 to the intercalated disc (34,36) as well as to de novo gap junctions in epithelial cells (57) and endometrial cells (58). The mechanism of GJA1-20k trafficking appears to involve regulation of cytoskeleton organization (36).…”

“…In other studies, it has been identified that GJA1-20k is necessary for the trafficking of full-length Cx43 to the intercalated disc (34,36) as well as to de novo gap junctions in epithelial cells (57) and endometrial cells (58). The mechanism of GJA1-20k trafficking appears to involve regulation of cytoskeleton organization (36). GJA1-20k may be a potent scaffolding protein, organizing the interaction among cytoskeleton, membrane, and vesicles containing Cx43 channel (34) or organelles such as mitochondria (37).…”

“…For instance, in addition to inducing constriction of the mitochondrial membrane, leading to subsequent Drp1-mediated fission (55), it is possible for the actin cytoskeleton to create contractile rings resulting in mitochondrial fission by sheer force generation, independent of Drp1 activity (54). Thus, via regulating the actin cytoskeleton (36) and associating with the OMM (Figure 3E), GJA1-20k may still affect mitochondrial fission by helping tether actin to the OMM. It would be interesting in future studies to decipher such mechanisms underlying the interplay among GJA1-20k, the actin cytoskeleton, and mitochondrial division.…”

“…Given the tropism of GJA1-20k to cardiac mitochondria, we were interested in examining the effect of exogenous GJA1-20k on mitochondrial quantity and morphology in the heart. To do so, we introduced GST-GFP control or GJA1-20k-GFP into the mouse heart in vivo by recombinant AAV9 gene transfer at 3 × 10 10 vector genomes per mouse at 4 weeks prior to sacrifice (36). Expression of GFP-tagged proteins at 4 weeks after AAV9 injection was confirmed by immunofluorescence imaging of heart tissue, as shown in Figure 4A.…”

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

“…It is also known as chaperone protein. It appears in Golgi apparatus earlier than GJA1‐Full Length (Basheer et al, ; Basheer & Shaw, ), and has obvious mitochondrial properties (Basheer, Fu, Shimura, et al, ). So we speculate that GJA1‐20 k may involve in the process of transporting functional GJA1 to the mitochondria.…”