Abstract Body: Introduction:
Doxorubicin (DOX) is an effective anticancer agent, but its use is limited by dose-dependent cardiotoxicity. AMP-activated protein kinase (AMPK) is a critical metabolic regulator in cardiac cells known for its cardioprotective effects, potentially mediated by regulating autophagy and mitophagy to clear damaged cellular components. AMPK functions as a heterotrimeric complex with catalytic alpha and regulatory beta and gamma subunits, each existing as multiple isoforms (α1/2, β1/2, γ1/2/3) that exert distinct biological functions. While AMPK α2 knockout mice show altered responses to DOX, the specific roles of the γ1 and γ2 isoforms remain unclear. This study investigates whether these isoforms modulate autophagy to protect against DOX-induced damage in H9C2 cardiomyoblast cells.
Methods:
H9C2 cells were transfected with siRNA to knock down AMPK γ1 and γ2, with silencing efficiency confirmed by Western Blot. Autophagic flux was assessed by measuring LC3B-II levels via Western Blot in cells treated with DOX (1μM, 16h), with or without lysosomal inhibitors. Confocal microscopy using an mRFP-GFP-LC3B tandem reporter was employed to visualize autophagosomes and autolysosomes. Cell viability was quantified using Propidium Iodide (PI) staining, with Hoechst 33342 staining for normalization.
Results:
siRNA transfection successfully reduced AMPK γ1 and γ2 protein expression by over 50%. Knockdown of either isoform significantly impaired autophagic flux, as evidenced by reduced LC3B-II turnover on Western Blots and a marked decrease in autophagosome puncta on confocal microscopy. This autophagic deficiency was accompanied by increased vulnerability to DOX, with PI staining showing significantly exacerbated cardiomyocyte death in both knockdown groups compared to controls.
Conclusion:
Our findings demonstrate that both AMPK γ1 and γ2 isoforms are essential for maintaining autophagic flux in H9C2 cardiomyoblasts. Loss of either subunit not only impairs autophagy but also sensitizes cells to DOX-induced injury. We conclude that AMPK γ1 and γ2 are both required to limit DOX cardiotoxicity by sustaining efficient autophagic clearance.