Targeting a Calcium‐Associated Mitochondrial Vulnerability in TET2‐Deficient Myeloid Cells

Figures

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  FIGURE 1. Acute TET2‐knockdown macrophage co‐culture modelling in CDMs reveals cardiac injury phenotypes. (A) Schematic of the TET2‐KD macrophage‐CDM co‐culture model. Monocytes were differentiated into macrophages (MΦs) using phorbol 12‐myristate 13‐acetate (PMA), transfected with scrambled siRNA (siScr) or siRNA targeting TET2 (siTET2), labelled with Qtracker, and co‐cultured with CDMs for 48 h to allow infiltration. (B) Two‐dimensional views of CDMs infiltrated by siScr or siTET2 macrophages. Nuclei were stained with DAPI (blue), cardiomyocytes with α‐actinin (green), exogenously added macrophages with Qtracker (magenta), and macrophages with CD68 (red). Scale bars, 100 µm; inset, 50 µm. (C) Three‐dimensional reconstructions of CDMs showing α‐actinin‐positive cardiomyocytes and Qtracker‐labelled macrophages. Scale bar, 100 µm. (D, E) Quantification of total (D) and average (E) macrophage‐infiltrated areas in CDMs as in (B). n = 3 biologically independent experiments per condition; more than 30 regions of interest from multiple organoids were analysed across the three replicates, with region‐level measurements averaged within each biological replicate to generate a single replicate‐level…

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  FIGURE 2. Chemical‐genetic screening identifies Lanatoside C as a selective inhibitor of TET2‐KD macrophages. (A) Screening workflow for identifying compounds preferentially targeting TET2‐KD macrophages. siScr and siTET2 THP‐1‐derived macrophages were plated in 384‐well format and treated with 489 compounds from the TargetMol Anti‐Cardiovascular Disease Compound Library for 48 h. Viability was measured by Alamar Blue and normalized to genotype‐matched DMSO controls. Hits were selected based on preservation of siScr viability > 80% and reduction of siTET2 viability < 70%, followed by secondary validation and prioritization of Lanatoside C. (B) Heatmap of relative cell viability in siScr or siTET2 macrophages treated with 489 cardiovascular disease‐related bioactive compounds (10 µM, 48 h). Viability was assessed by Alamar Blue and expressed relative to the corresponding genotype‐matched vehicle controls. n = 4 biologically independent replicates per treatment. (C) Quantification of cell viability by Alamar Blue assay in siScr or siTET2 macrophages treated with vehicle or a subset of 8 selected compounds. n = 8 biologically independent replicates per condition. (D) Quantification of…

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  FIGURE 3. Lanatoside C selectively induces apoptosis in TET2‐knockdown macrophages through calcium‐associated mitochondrial stress. (A) Schematic representation of proposed pathways involved in Lanatoside C‐induced apoptosis in siTET2 macrophages. (B) Heatmap of Na+/K+ pump and Na+/Ca2+ exchanger gene expression (ATP1A1‐3, ATP1B1‐3, SLC8A1‐2) from bulk RNA sequencing of siScr or siTET2 macrophages treated with vehicle or Lanatoside C. n = 3 biologically independent replicates per condition. (C) Quantification of intracellular Ca2+‐sensitive Cal‐520 fluorescence in siScr and siTET2 macrophages treated with vehicle or Lanatoside C. Fluorescence values were normalized to the siScr vehicle group. n = 8 biologically independent replicates per condition. (D) Quantification of JC‐10 red/green fluorescence ratio in siScr and siTET2 macrophages treated with vehicle or Lanatoside C. n = 8 biologically independent replicates per condition. (E) Representative JC‐10 fluorescence images of siScr and siTET2 macrophages treated with vehicle or Lanatoside C, corresponding to the quantification shown in (D). Red fluorescence indicates JC‐10 J‐aggregates in polarized mitochondria, whereas green fluorescence…

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  FIGURE 4. Lanatoside C attenuates cardiac injury phenotypes in TET2‐KD macrophage‐infiltrated CDMs. (A) Relative IL‐1β, IL‐6, and IL‐18 levels in the culture medium of CDMs infiltrated by siScr or siTET2 macrophages and treated with vehicle or Lanatoside C (10 µM, 48 h), measured by ELISA and normalized to the siScr Vehicle group. n = 8 biologically independent replicates per condition. (B) Quantification of cleaved Caspase‐3‐positive areas in CDMs infiltrated by siScr or siTET2 macrophages treated with vehicle or Lanatoside C. n = 8 biologically independent samples per condition. (C,D) Representative immunofluorescence images of CDMs stained with α‐actinin (green) and cleaved‐Caspase‐3 (magenta) after treatment as in (B). Scale bar, 10 µm. (E) Quantification of cardiac troponin T (cTnT) release into culture media of CDMs infiltrated by siScr or siTET2 macrophages treated with vehicle or Lanatoside C. n = 4 biologically independent replicates per condition. (F) Representative calcium flux transients in CDMs infiltrated by siScr or siTET2 macrophages treated with vehicle or Lanatoside C. (G,H) Quantification of beating frequency (G) and amplitude (H) in CDMs infiltrated by siScr or siTET2…

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  FIGURE 5. Lanatoside C attenuates Tet2‐deficient donor‐derived myeloid representation in a partial donor‐cell reconstitution model. (A) Schematic of the bone marrow transplantation strategy. Bone marrow cells from either Tet2+/+ or Tet2−/− CD45.2+ donor mice were transplanted separately into irradiated CD45.1+ recipient mice. Conditioning consisted of 11 Gy total‐body irradiation in two 5.5‐Gy fractions separated by 4 h. Each recipient received 5 × 106 donor cells intravenously per day for three consecutive days. Week 0 denotes irradiation and initiation of the three‐day transplantation schedule. At week 4, baseline peripheral‐blood donor chimerism was assessed before treatment allocation. Vehicle or Lanatoside C was then administered daily for one month (approximately weeks 4–8), with endpoint peripheral‐blood and cardiac‐tissue collection at approximately week 8. The experiment achieved partial donor reconstitution, consistent with incomplete marrow ablation inferred from blood chimerism. (B) Representative flow‐cytometry plots showing CD45.2+ donor‐derived leukocytes among circulating live CD45+ white blood cells at four weeks after transplantation, before treatment initiation, in…