Role of Bcl2-associated Athanogene 3 in Turnover of Gap Junction Protein, Connexin 43, in Neonatal Cardiomyocytes

Farzaneh Ghasemi Tahrir,Valerie Myers,Joseph Y Cheung,Manish Gupta,Jennifer Gordon,Arthur M Feldman,Kamel Khalili

doi:10.1038/s41598-019-44139-w

Farzaneh Ghasemi Tahrir, Valerie Myers + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-44139-w

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Journal: Scientific Reports	Publication Date: May 21, 2019
Citations: 13	License type: open-access

Affiliation: Temple University

Abstract

Any pathological stress that impairs expression, turnover and phosphorylation of connexin 43 (Cx43), one of the major proteins of gap junctions, can adversely impact myocardial cell behavior, thus leading to the development of cardiac arrhythmias and heart failure. Our results in primary neonatal rat ventricular cardiomyocytes (NRVCs) show that impairment of the autophagy-lysosome pathway dysregulates degradation of Cx43, either by inhibiting lysosomal activity or suppressing the level of Bcl2-associated athanogene 3 (BAG3), a stress-induced pleiotropic protein that is involved in protein quality control (PQC) via the autophagy pathway. Inhibition of lysosomal activity leads to the accumulation of Cx43 aggregates and suppression of BAG3 significantly diminished turnover of Cx43. In addition, knock-down of BAG3 reduced the levels of Cx43 by dysregulating Cx43 protein stability. Under stress conditions, expression of BAG3 affected the state of Cx43 phosphorylation and its degradation. Furthermore, we found that BAG3 co-localized with the cytoskeleton protein, α-Tubulin, and depolymerization of α-Tubulin led to the intracellular accumulation of Cx43. These observations ascribe a novel function for BAG3 that involves control of Cx43 turnover under normal and stress conditions and potentially for optimizing communication of cardiac muscle cells through gap junctions.

Highlights

Bcl2-associated athanogene 3 (BAG3) is a 575-amino acid protein that is highly expressed in cardiac and skeletal muscle[13]
In order to investigate whether connexin 43 (Cx43) degradation in neonatal rat ventricular cardiomyocytes (NRVCs) occurs through lysosomes, lysosomal activity was inhibited by treating the cardiomyocytes with 50 nM Bafilomycin A1 (BafA1) for 3 hours and the levels of the autophagy markers, LC3 I and LC3 II, and Cx43 were analyzed by Western blot
We have identified the importance of BAG3 in regulating turnover of Cx43 under normal, as well as stress conditions, in primary cultures of neonatal cardiomyocytes using several complementary approaches

Summary

Introduction

BAG3 is a 575-amino acid protein that is highly expressed in cardiac and skeletal muscle[13]. BAG3 deficiency results in myofibrillar degeneration followed by the development of lethal cardiomyopathy and death by 4 weeks of age[19]. While the role of BAG3 in quality control has been reported by several researchers, to our knowledge, there is no study describing the impact of BAG3 on gap junction turnover, either in basal or stress conditions. We used primary cultures of NRVCs and found that there was a significant reduction in Cx43 abundance as well as Cx43 turnover in BAG3-suppressed cardiomyocytes. We found that suppression of BAG3 resulted in destabilization of Cx43. Our results showed that BAG3 colocalized with the tubulin network and tubulin depolymerization led to significant accumulation of Cx43 within cardiomyocytes

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