CK2α Deficiency Drives Myocardial Fibrosis via Desmin‐Induced Mitochondrial Dysfunction

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  FIGURE 1. Downregulation of cardiomyocyte CK2α correlates with myocardial fibrosis. (a,b) Bioinformatic analysis showing reduced CK2α mRNA expression in human fibrotic hearts (a) and murine cardiac fibrosis models (b) based on the indicated GEO datasets. HCM, Hypertrophic cardiomyopathy; DCM, Dilated cardiomyopathy; ICM, ischemic cardiomyopathy; TAC, transverse aortic constriction; MI, myocardial infarction; SHR, spontaneously hypertensive rat; ISO, isoproterenol. (c,d) Representative Masson's trichrome staining (c) and immunohistochemistry (IHC) staining of CK2α (d) in heart sections from Sham and MI mice. (e) Representative immunofluorescence images showing the localization and expression of CK2α (red) and α‐Actinin (green) in Sham and MI mouse hearts. (f) Representative immunoblots (left) and quantification (right) of CK2α and fibrosis markers in the infarct border zone from Sham and MI mice (n = 3 per group). (g) RT‐qPCR analysis of CK2α and fibrosis‐related genes (α‐SMA, Col1a1, and Col3a1) in Sham (n = 6) and MI (n = 4) mouse hearts. (h,i) Representative Masson's trichrome staining (h) and IHC staining of CK2α (i) in heart sections from Saline‐ and ISO‐treated mice. (j)…

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  FIGURE 2. Cardiomyocyte CK2α deficiency promotes cardiac fibroblast activation through paracrine signaling. (a) Heatmap analysis of differentially expressed genes related to extracellular matrix (ECM) and profibrotic factors in H9c2 cells from the E‐MTAB‐8067 dataset following CK2α knockdown (shCK2α). (b) Representative immunoblotting images (left) and relative quantitative analysis (right) of α‐SMA, Collagen 1, Collagen 3, and CK2α in H9c2 cells transfected with siNTC, siCK2α‐1, or siCK2α‐2 (n = 3 independent biological replicates). (c) RT‐qPCR analysis of CK2α, α‐SMA, Col1a1, and Col3a1 mRNA levels in primary cardiomyocytes (CMs) after CK2α knockdown (n = 3 independent biological replicates). (d) Schematic diagram illustrating the experimental design: primary cardiomyocytes (CMs) were treated with siRNA, and the resulting conditional medium (CM) was collected to treat primary cardiac fibroblasts (CFs). (e) Relative cell viability of CFs treated with conditional medium from siNTC‐ or siCK2α‐transfected CMs, determined by CCK‐8 assay (n = 3 independent technical replicates). (f) Representative images (left) and quantification (right) of the wound healing assay in CFs treated with the…

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  FIGURE 3. Cardiomyocyte‐specific CK2α deficiency drives age‐dependent fibrosis and cardiac dysfunction. (a) Schematic illustrating the generation of inducible cardiomyocyte‐specific CK2α knockout mice (CK2αcKO, Myh6‐Cre/CK2αfl/fl) via tamoxifen (50 mg/kg via intraperitoneal injection for 5 days) administration at 1 month of age. (b) Representative Masson's trichrome staining of left ventricular (LV) sections from Control (CTL) and CK2αcKO mice at 3, 12, and 20 months. Scale bar: 100 µm. Right: Quantification of fibrotic area showing progressive collagen deposition (n = 5 per group). (c) Immunoblot and quantitative analysis of fibrotic markers (Collagen 1, Collagen 3, α‐SMA, TGF‐β1) in 20‐month‐old hearts (n = 4 per group). (d) Immunohistochemical staining and quantification of Collagen 1‐positive cells in 20‐month‐old hearts (n = 5 per group). (e) Representative immunoblotting images (left) and relative quantitative analysis (right) of Collagen 1, Collagen 3, α‐SMA, and CK2α in primary cardiomyocytes (CMs) and cardiac fibroblasts (CFs) isolated from CTL and CK2αcKO mice (n = 3 independent technical replicates). (f) Echocardiographic assessment of cardiac function in aged mice (20 months),…

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  FIGURE 4. CK2α deficiency triggers mitochondrial proteome collapse and induces a global bioenergetic crisis. (a) Subcellular localization analysis of differentially expressed proteins (DEPs) in left ventricular tissue from CTL and CK2αcKO mice. The pie chart highlights the specific depletion of the mitochondrial proteome (red outline), with 107 proteins significantly downregulated. (b) KEGG pathway enrichment analysis showing the downregulated pathways in CK2αcKO hearts, predominantly involving oxidative phosphorylation, the TCA cycle, and fatty acid/BCAA metabolism. (c) Heatmap visualization of DEPs related to metabolic pathways. Red indicates upregulation (mainly glycolysis); blue indicates downregulation (FAO, BCAA degradation, and TCA cycle). (d) Representative Oil Red O staining images of ventricular sections in 20‐month‐old hearts. Scale bar: 50 µm. (e–g) Quantification of cardiac metabolites in 20‐month‐old hearts (n = 4 or 5 per group): L‐lactate content (e), branched‐chain amino acids (BCAAs) content (f), and TCA cycle intermediates succinate and α‐ketoglutarate content (g). (h) Heatmap displaying the widespread downregulation of electron transport chain (ETC) subunits across…

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  FIGURE 5. CK2α deficiency induces mitochondrial damage and promotes cardiac fibrosis. (a) Representative transmission electron microscopy (TEM) images of left ventricular myocardium in 20‐month‐old hearts. Scale bar: 2 µm (left) and 1 µm (zoom, right). (b) Quantification of individual mitochondrial area and cristae density from TEM images (n = 3 per group). (c) Representative dihydroethidium (DHE) staining (red) in 20‐month‐old hearts. Scale bar: 30 µm. (d) Measurement of myocardial MDA content (lipid peroxidation marker) and SOD activity in 20‐month‐old hearts (n = 4 per group). (e,f) Representative immunoblots (e) and quantification (f) of stress signaling (P‐p38) and mitochondrial antioxidant proteins (SOD2, SIRT3) in 20‐month‐old hearts (n = 3 per group). (g) The relative abundance of mtDNA (ND1 and ND4) leaked into the cytosol was quantified by RT‐qPCR in cardiac tissue, with the nuclear gene Tert serving as the reference control (n = 4 per group). (h) Western blot and relative quantification analysis of p65 NF‐κB distribution in cytoplasmic and nuclear fractions. Lamin B1 and GAPDH were used as nuclear and cytoplasmic loading controls, respectively (n = 3 per group). (i) RT‐qPCR…

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  FIGURE 6. CK2α prevents mitochondrial collapse and fibrosis by phosphorylating Desmin at Thr452 to maintain protein quality control. (a) Venn diagram illustrating the identification of Desmin (encoding by Des) and Cryab by intersecting downregulated mitochondrial phosphoproteins in CK2αcKO hearts with fibrosis‐related datasets. (b, c) Co‐immunoprecipitation (Co‐IP) assays confirming the interaction between CK2α and Desmin. (b) Endogenous interaction in H9c2 cell lysates. (c) Exogenous interaction in HEK293T cells co‐transfected with Flag–Desmin and HA‐CK2α. (d) GST‐pulldown assay using purified recombinant His‐CK2α and GST‐Desmin, confirming direct physical binding. (e) In vitro kinase assay detecting thiophosphate ester formation, showing direct phosphorylation of Desmin by CK2α. (f) Phosphoproteomic quantification of Desmin phosphorylation sites (Ser25, Thr452) in CTL and CK2αcKO hearts. (g) Immunoprecipitation of Flag‐Desmin (WT, S25A, T452A) from HEK293T cells co‐expressed with HA‐CK2α, followed by immunoblotting for total phosphorylation (p‐Ser/Thr). (h) In vitro kinase assay using purified Desmin mutants, identifying Thr452 as the direct CK2α phosphorylation site. (i) Co‐IP analysis…

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  FIGURE 7. AAV9‐mediated CK2α overexpression protects against isoproterenol‐induced cardiac fibrosis and mitochondrial dysfunction. (a) Schematic representation of the experimental design. Mice received AAV9‐cTNT‐CK2α (or AAV9‐NC) at 8 weeks of age, followed by isoproterenol (ISO, 50 mg/kg/day) or saline treatment from week 12 to 15. (b,c) Representative echocardiographic images (b) and quantification of ejection fraction (EF) and fractional shortening (FS) (c). (d) Serum analysis of cardiac injury markers BNP and CK‐MB. (e,f) Gross morphology of whole hearts (e) and quantification of the heart‐weight‐to‐body‐weight (HW/BW) ratio (f). Scale bar: 2 mm. (g,h) Representative Masson's trichrome staining (g) and quantification of fibrotic area (h). Scale bar: 20 µm. (i,j) Representative TEM images (i) and quantification of mitochondrial cristae density (j). Scale bar: 2 µm. (k) Measurement of cardiac ATP content in cardiac tissue lysates. (l) Analysis of oxidative stress markers in cardiac tissue lysates: MDA content (left) and SOD activity (right). (m) Immunoblot analysis of ETC complex subunits (CI–CV) in heart lysates (n = 3 per group). (n) Immunoblot analysis of fibrotic markers (Collagen 1,…