Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury
Wassim A. Basheer, … , TingTing Hong, Robin M. Shaw
Wassim A. Basheer, … , TingTing Hong, Robin M. Shaw
Published October 18, 2018
Citation Information: JCI Insight. 2018;3(20):e121900. https://doi.org/10.1172/jci.insight.121900.
View: Text | PDF
Research Article Metabolism Muscle biology

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

Abstract

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.

Authors

Wassim A. Basheer, Ying Fu, Daisuke Shimura, Shaohua Xiao, Sosse Agvanian, Diana M. Hernandez, Tara C. Hitzeman, TingTing Hong, Robin M. Shaw

×

Figure 3

GJA1-20k targets mitochondria after I/R injury and associates with the OMM.

Options: View larger image (or click on image) Download as PowerPoint
GJA1-20k targets mitochondria after I/R injury and associates with the O...
(A) Confocal images of mouse heart cryosections following I/R injury. Tissue is costained with a Cx43 N-terminus antibody (Cx43-NT, red) and a Cx43 C-terminus antibody (Cx43-CT, green), and cell membranes are labeled with WGA (white dotted line). Scale bar: 10 μm. Pearson’s coefficient for the colocalization signal (yellow, merged images) is quantified in B. Data are mean ± SEM (number of cells quantified is shown on the graph). ****P < 0.0001, unpaired t test. (C) Biochemical fractionation of isolated hearts after I/R, showing GJA1-20k protein distribution in the supernatant prior to ultracentrifugation (F1) versus mitochondrial fractions (F2, F3). Low- and high-exposure blots of GJA1-20k are shown as well as mitochondrial marker (Tom20) and plasma membrane markers (Na+/K+ATPase and N-cadherin). (D) 3D STORM images of a single cardiac mitochondrion (outlined with a blue line) expressing V5-tagged GJA1-20k (green). Tom20 is shown in red. Scale bar: 0.5 μm. (E) Western blot of mitochondrial fractions isolated from HEK cells transfected with GFP-tagged GJA1-20k and subjected to a proteinase K protection assay (1 μg/ml PK on ice for 30 minutes). PK treatment completely abolished GJA1-20k at the outer mitochondrial membrane (OMM), as assessed using GFP and Cx43-CT antibodies. Tom20 is used as an OMM marker and CoxIV is used as an inner mitochondrial membrane marker.