docling/tests/data/groundtruth/docling_v2/research.0509.nxml.itxt

item-0 at level 0: unspecified: group _root_
  item-1 at level 1: section_header: Introduction
    item-2 at level 2: text: Cancer therapeutics have made re ... eding risk resulting from its use [7].
    item-3 at level 2: text: Chemotherapy-induced cardiotoxic ...  the onset and progression of AF [11].
    item-4 at level 2: text: The mitochondrial quality survei ... alance of mitochondrial dynamics [17].
    item-5 at level 2: text: A previous study found that PI3K ... pathogenesis of ibrutinib-mediated AF.
  item-6 at level 1: section_header: Ibrutinib increases AF susceptib ... d promotes mitochondrial fragmentation
    item-7 at level 2: text: To assess the proarrhythmic pote ... nce of atrial mitochondria (Fig. S1I).
  item-8 at level 1: section_header: Ibrutinib induces AF by impairin ... ction and structure of atrial myocytes
    item-9 at level 2: text: We investigated the molecular me ...  mitochondrial structure and function.
  item-10 at level 1: section_header: Ibrutinib disrupts mitochondrial ... own-regulating atrial AKAP1 expression
    item-11 at level 2: text: As GO and KEGG enrichment analys ... ed expression of DRP1 (Fig. 2D and E).
    item-12 at level 2: text: We computed the activity score o ... between ibrutinib and AKAP1 (Fig. 2J).
  item-13 at level 1: section_header: AKAP1 down-regulation mediates DRP1 dephosphorylation in HL-1 myocytes
    item-14 at level 2: text: We investigated the interaction  ... ondrial fission marker (Fig. 3B to F).
  item-15 at level 1: section_header: Ibrutinib-induced AKAP1 depletio ... olarization via DRP1 dephosphorylation
    item-16 at level 2: text: Mitochondrial dynamic imbalance  ... l morphology to normal (Fig. 4A to E).
    item-17 at level 2: text: Western blot analysis showed tha ...  AKAP1 overexpression (Fig. 4O and P).
  item-18 at level 1: section_header: AKAP1 deficiency impairs mitocho ... tabolic reprogramming in HL-1 myocytes
    item-19 at level 2: text: Mitochondrial bioenergetics and  ... rial fission inhibitor (Fig. 5A to F).
    item-20 at level 2: text: Impaired mitochondrial respirati ... -treated with Mdivi-1 (Fig. 5L and M).
  item-21 at level 1: section_header: AKAP1 down-regulation is involve ... is, oxidative stress, and inflammation
    item-22 at level 2: text: Insufficient energy production c ... r Mdivi-1 intervention (Fig. 6A to C).
    item-23 at level 2: text: We next elucidated the regulator ...  Mdivi-1 intervention (Fig. 6F and G).
    item-24 at level 2: text: Mitochondrial dysfunction not on ... i-1 cotreatment groups (Fig. 6I to K).
  item-25 at level 1: section_header: AKAP1 overexpression prevents ib ... S and restoring mitochondrial function
    item-26 at level 2: text: To verify whether AKAP1 up-regul ... y AKAP1 overexpression (Fig. 7F to I).
    item-27 at level 2: text: MitoSOX staining revealed that m ...  inflammatory response (Fig. 7L to P).
  item-28 at level 1: section_header: Discussion
    item-29 at level 2: text: Mitochondrial damage serves as a ... t for exploring preventive approaches.
    item-30 at level 2: text: AKAP1, a scaffolding protein, an ... sturbances through off-target effects.
    item-31 at level 2: text: Recent studies have investigated ...  pathogenesis of ibrutinib-induced AF.
    item-32 at level 2: text: Our study utilized high-throughp ... xorubicin-induced cardiotoxicity [27].
    item-33 at level 2: text: Excessive fission depletes ATPs  ... forms a pathological substrate for AF.
    item-34 at level 2: text: In this study, we discovered tha ... oxicity of tyrosine kinase inhibitors.
    item-35 at level 2: text: Increasing evidence suggests tha ... ators for AKAP1 are not yet available.
  item-36 at level 1: section_header: Animal model
    item-37 at level 2: text: C57BL/6J mice (8 to 10 weeks, we ... d libitum throughout the study period.
  item-38 at level 1: section_header: Cell culture and lentivirus transfection
    item-39 at level 2: text: For stable AKAP1 overexpression, ... ailed in the related prior study [53].
  item-40 at level 1: section_header: In vivo electrophysiology study
    item-41 at level 2: text: Electrophysiological studies on  ... pisodes of each animal was documented.
  item-42 at level 1: section_header: Echocardiography
    item-43 at level 2: text: The Vevo 2100 Imaging System (FU ... ted the echocardiographic examination.
  item-44 at level 1: section_header: Transmission electron microscopy
    item-45 at level 2: text: Retrograde aortic perfusion with ... s described in our previous study [21]
  item-46 at level 1: section_header: Single-cell RNA-seq analysis
    item-47 at level 2: text: The single-cell transcriptome da ...  pathway activities of specific cells.
  item-48 at level 1: section_header: Confocal imaging
    item-49 at level 2: text: Fluorescent images were obtained ... ntibodies used for immunofluorescence.
    item-50 at level 2: text: MitoTracker Red (0.5 μM, 30 min) ... he MitoSOX Red (5 μM, 20 min) Reagent.
  item-51 at level 1: section_header: Isolation of primary atrial myocytes and atrial mitochondria
    item-52 at level 2: text: Atrial myocytes were isolated us ... anual (Thermo Fisher Scientific, USA).
  item-53 at level 1: section_header: Western blot analysis
    item-54 at level 2: text: Atrial tissues or cultured cell  ... the antibodies documented in Table S1.
  item-55 at level 1: section_header: mtDNA copy number and quantitative PCR
    item-56 at level 2: text: The mitochondrial DNA copy numbe ...  analysis of the respective sequences.
  item-57 at level 1: section_header: Figures
  item-59 at level 1: picture
    item-59 at level 2: caption: Fig. 1. Ibrutinib induces atrial fibrillation (AF) by impairing mitochondrial function and structures of atrial myocytes. (A and B) Expression heatmap and volcano map of differentially expressed atrial proteins, illustrating that 170 proteins were up-regulated and 208 were down-regulated in ibrutinib-treated mice compared with control mice. N = 3 independent mice per group. (C to H) Gene Ontology and KEGG enrichment analysis of the 378 differentially expressed proteins in atrial tissue. (I to L) GSEA of differentially expressed proteins following ibrutinib treatment.
  item-61 at level 1: picture
    item-61 at level 2: caption: Fig. 2. Ibrutinib disrupts mitochondrial homeostasis by down-regulating atrial AKAP1 expression. (A) Volcano plot labeled with the top mitochondria-related differentially expressed proteins. (B) Expression of marker genes for cell-type annotation is indicated on the DotPlot. (C) Uniform manifold approximation and projection representation of all single cells color-coded for their assigned major cell type. (D and E) Featureplots showing the expression patterns of AKAP1 and DRP1 in atrial tissues from AF and control groups. (F to H) Analysis of the correlation between AKAP1 expression and activity score of mitochondria-related pathways. (I) Atrial pan-subpopulation correlation analysis of AKAP1 expression and OXPHOS pathway activity. (J) Molecular docking analysis of the interaction between ibrutinib and AKAP1.
  item-63 at level 1: picture
    item-63 at level 2: caption: Fig. 3. AKAP1 binding mediates DRP1 phosphorylation in atrial myocytes. (A) Immunofluorescence imaging of AKAP1 and DRP1 in ibrutinib-treated atrial myocytes. (B to F) Proteins were isolated from atrial myocytes. Western blotting was used to assess AKAP1, DRP1, FIS1, and phosphorylated DRP1 expression levels. ***P < 0.001. ns, not significant.
  item-65 at level 1: picture
    item-65 at level 2: caption: Fig. 4. Ibrutinib-induced AKAP1 depletion promotes mitochondrial fission and membrane depolarization via DRP1 dephosphorylation. (A to E) MitoTracker staining for labeling mitochondria in atrial myocytes in vitro. Average mitochondrial length, ratio of fragmented/tubular mitochondria, and mitochondrial morphological parameters were determined. (F to N) Western blot analysis of mitochondrial DRP1 (mito-DRP1), total DRP1 (t-DRP1), phosphorylated DRP1, FIS1, MFN1, and OPA1 in HL-1 cells. VDAC1 was used as a loading control for mitochondrial proteins. (O) Red-to-green ratio of JC-1 fluorescence intensity. (P) Atrial myocytes loaded with JC-1 to analyze changes in mitochondrial membrane potential. N = 8 independent cell samples per group. **P < 0.01, ***P < 0.001.
  item-67 at level 1: picture
    item-67 at level 2: caption: Fig. 5. AKAP1 deficiency impairs mitochondrial respiratory function and promotes metabolic reprogramming in atrial myocytes. (A to F) Analysis of HL-1 mitochondrial bioenergetics using the Seahorse XFe96 Analyzer. OCR measurements were taken continuously from baseline and after the sequential addition of 2 mM oligomycin, 1 mM FCCP, and 0.5 mM R/A to measure basal respiration, maximal respiration, spare respiratory capacity, proton leak, and ATP-production levels. (G to J) Total and individual rates of ATP production as mediated by glycolysis or mitochondrial metabolism in HL-1 cells transduced with control and LV-Akap1. (K) Ratio between ATP produced by OXPHOS and that by glycolysis in HL-1 cells transduced with control and LV-Akap1. (L and M) ELISA analysis of mitochondrial respiration complex I/III activities. N = 8 independent cell samples per group. *P < 0.05, **P < 0.01, ***P < 0.001.
  item-69 at level 1: picture
    item-69 at level 2: caption: Fig. 6. AKAP1 down-regulation in impaired mitochondrial biogenesis, oxidative stress, and inflammation. (A to C) qPCR analysis of mitochondrial biogenesis parameters (mtDNA copy number, PGC-1α, and NRF1). (D and E) ELISA analysis of redox balance biomarkers, including MDA and GSH contents. (F and G) MitoSOX staining of mitochondrial ROS in HL-1 cells. (H) Visual GSEA of inflammation response pathway in ibrutinib-treated myocytes. (I to K) ELISA of atrial inflammatory biomarkers, including TNF-α, IL-6, and IL-18. N = 8 independent cell samples per group. *P < 0.05, **P < 0.01, ***P < 0.001.
  item-71 at level 1: picture
    item-71 at level 2: caption: Fig. 7. AKAP1 overexpression prevents ibrutinib-induced AF by improving MQS and restoring mitochondrial function. (A) Simultaneous recordings of surface ECG following intraesophageal burst pacing. (B) Quantification of AF time. (C) AF inducibility in control and ibrutinib-treated mice with and without AAV-Akap1 transfection. (D and E) Myocardial fibrosis was assessed in each group through Sirius Red staining. The proportion of fibrotic tissue to myocardial tissue was quantified for each group. (F to I) Western blot analysis of total DRP1 (t-DRP1), phosphorylated DRP1, MFN1, and NRF1 in atrial tissues. (J and K) Images and quantification of isolated atrial myocytes loaded with the mitochondrial ROS indicator MitoSox Red. (L to P) ELISA of MQS/redox/inflammation biomarkers, including complex I/III, MDA, GSH, and IL-18. N = 8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001.
  item-73 at level 1: picture
    item-73 at level 2: caption: Fig. 8. This schematic illustrates the proposed mechanism by which ibrutinib promotes atrial fibrillation (AF). Ibrutinib treatment leads to the downregulation of A-kinase anchoring protein 1 (AKAP1), triggering mitochondrial quality surveillance (MQS) impairment. This results in enhanced translocation of dynamin-related protein 1 (DRP1) to the mitochondria, driving mitochondrial fission. Concurrently, metabolic reprogramming is characterized by increased glycolysis and decreased oxidative phosphorylation (OXPHOS), alongside reduced mitochondrial biogenesis. These mitochondrial dysfunctions elevate oxidative stress and inflammatory responses, contributing to atrial metabolic and structural remodeling, ultimately leading to ibrutinib-induced AF.
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