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

Abstract

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 (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. Results Compared 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. 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.

Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa)

“…To the best of our knowledge, there are no previous data demonstrating a regulatory role of Zfhx3 on Nav1.5 channels. Conversely, data showing its effects on the expression and function of cardiac K + channels are more abundant [ 4 , 27 , 28 ]. It was shown that Zfhx3 silencing in HL-1 cells increased the expressions of Kv1.4, Kv1.5 and Kir3.4 channels, which resulted in augmented ultra-rapid delayed rectifier (I Kur ), transient outward (I to ) and acetylcholine-sensitive potassium (I KAch ) currents and the shortening of the action potential duration [ 27 ].…”

“…It was shown that Zfhx3 silencing in HL-1 cells increased the expressions of Kv1.4, Kv1.5 and Kir3.4 channels, which resulted in augmented ultra-rapid delayed rectifier (I Kur ), transient outward (I to ) and acetylcholine-sensitive potassium (I KAch ) currents and the shortening of the action potential duration [ 27 ]. More recently, Lkhagva et al described that Zfhx3 silencing in HL-1 cells led to a significant increase in the ATP-sensitive K current (I KATP ) [ 28 ]. There are also data suggesting that Zfhx3 regulates intracellular calcium handling, since Zfhx3 knockdown in HL-1 cells increased the ryanodine receptor ( RYR2 ), RyR2 p2808, and SERCA2a ( ATP2A2 ) mRNA expression [ 27 ].…”

Illuminating physiology

“…Today, bioluminescence-based experimental techniques using enzymatically active luciferin are widely available, aiding molecular physiology in fields as diverse as cardiovascular research, pancreatic ß cell synchronization or general cellular physiology. [4][5][6] From the simplest patches of photoreceptor protein in unicellular animals to complex compound eyes, manifold organisms develop various photoreceptors. This occurs in simple to highly complex species, in close adaptation to the environment and its demands.…”

“…Animals emit light to lure prey or potential mates, or to hide by camouflage and mimicry. Today, bioluminescence‐based experimental techniques using enzymatically active luciferin are widely available, aiding molecular physiology in fields as diverse as cardiovascular research, pancreatic ß cell synchronization or general cellular physiology 4–6 …”

Epigenetic Markers Are Associated With Differences in Isocyanate Biomarker Levels in Exposed Spray-Painters

“…ZFHX3 and FZD9 are two other genes associated with significant CpGs that have potentially important functions, including ZFHX3's involvement in regulating both intracellular calcium homeostasis (Zhao et al, 2019) and mitochondrial oxidative stress (Lkhagva et al, 2021), and FZD9's involvement in bone mineralization (Albers et al, 2011;Heilmann et al, 2013) andB-cell development (Ranheim et al, 2005). Bone mineralization is a pathway that also arose in our GWAS biomarker research (ACVR1, BMPRB) (Taylor et al, 2020) and has implications for calcium regulation.…”