["Acta Physiologica, Volume 231, Issue 4, April 2021. ", "\nAbstract\n\nAim\nTo 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).\n\n\nMethods\nThrough 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 (IKATP), 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.\n\n\nResults\nCompared with 1‐Hz pacing, 5‐Hz pacing increased ATP and ADP production, IKATP, 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 IKATP, 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.\n\n\nConclusion\nZFHX3 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.\n\n"]