ZFHX3 knockdown dysregulates mitochondrial adaptations to tachypacing in atrial myocytes through enhanced oxidative stress and calcium overload

Aim: To investigate the role of zinc finger homeobox 3 gene (ZFHX3) in tachypacing-induced mitochondrial dysfunction and explore its molecular mechanisms and potential as a therapeutic target in atrial fibrillation (AF). Methods: Through a bioluminescent assay, a patch clamp, confocal fluorescence and fluorescence microscopy, microplate enzyme activity assays and Western blotting, we studied ATP and ADP production, mitochondrial electron transfer chain complex activities, ATP-sensitive potassium channels (I_KATP), mitochondrial oxidative stress, Ca²⁺ content, and protein expression in control and ZFHX3 knockdown (KD) HL-1 cells subjected to 1 and 5-Hz pacing for 24 hours. Results: Compared with 1-Hz pacing, 5-Hz pacing increased ATP and ADP production, I_KATP, phosphorylated adenosine monophosphate-activated protein kinase and inositol 1,4,5-triphosphate (IP₃) receptor (IP₃R) protein expression. Tachypacing induced mitochondrial oxidative stress and Ca²⁺ overload in both cell types. Furthermore, under 1- and 5-Hz pacing, ZFHX3 KD cells showed higher I_KATP, ATP and ADP production, mitochondrial oxidative stress and Ca²⁺ content than control cells. Under 5-Hz pacing, 2-aminoethoxydiphenyl borate (2-APB; 3 μmol/L, an IP₃R inhibitor) and MitoTEMPO (10 µmol/L, a mitochondria-targeted antioxidant) reduced ADP and increased ATP production in both cell types; however, only 2-APB significantly reduced mitochondrial Ca²⁺ overload in control cells. Under 5-Hz pacing, mitochondrial oxidative stress was significantly reduced by both MitoTEMPO and 2-APB and only by 2-APB in control and ZFHX3 KD cells respectively. Conclusion: ZFHX3 KD cells modulate mitochondrial adaptations to tachypacing in HL-1 cardiomyocytes through Ca²⁺ overload, oxidative stress and metabolic disorder. Targeting IP₃R signalling or oxidative stress could reduce AF.