CACNB4 attenuates cardiac dysfunction by regulating calcium and ATP levels via interaction with RyR2

Figures

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  Fig. 1. Downregulation of CACNB4 in PBMCs from heart failure patients is associated with cardiac contractility. A, B Volcano plot showing differentially expressed genes in PBMCs from heart failure patients (GSE235757) compared to the normal group. Red dots represent upregulated genes, blue dots represent downregulated genes, and black dots represent non-significant genes. C, D KEGG pathway enrichment analysis of differentially expressed genes identified in PBMCs from heart failure patients. E, F Western blot analysis of CACNB4 protein expression in hypoxic myocardial cells and heart tissue from heart failure mice, respectively. **, p < 0.01; ***, p < 0.001. Results are from three or six repeated experiments

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  Fig. 2. Overexpression of CACNB4 alleviates cardiac function. A Western blot analysis of CACNB4 protein expression in heart tissue from sham, MI, and MI + OE–CACNB4 groups. B Echocardiography analysis of cardiac function indicators (LVIDd, LVIDs, EF%, FS%) in sham, MI, and MI + OE–CACNB4 groups. C Gross morphology of the heart from sham, MI, MI + OE–NC, and MI + OE–CACNB4 groups. D TTC staining showing infarct volume in sham, MI, and MI + OE–CACNB4 groups. E Hematoxylin and eosin (H&E) staining of heart tissue from sham, MI, MI + OE–NC, and MI + OE–CACNB4 groups (original magnification × 100 bars = 200 μm) (original magnification × 200 bars = 100 μm). F Masson staining showing collagen fiber deposition in heart tissue from sham (original magnification × 100 bars = 200 μm) (original magnification × 200 bars = 100 μm). G Western blot analysis of myocardial injury markers (TNI, BNP, and TNC) in sham, MI, MI + OE–NC, and MI + OE–CACNB4 groups. H ELISA analysis of ATP content in heart tissue from sham, MI, MI + OE–NC, and MI + OE–CACNB4 groups. I Fluo-3 staining showing Ca2+ levels in heart tissue from sham, MI, MI + OE–NC, and MI + OE–CACNB4 groups (original magnification × 200 bars = 100…

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  Fig. 3. Overexpression of CACNB4 enhances intracellular Ca2 + and ATP levels in cardiomyocytes. A Western blot analysis of CACNB4 protein expression in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–CACNB4 groups. B Western blot analysis of myocardial injury markers (TNI, α-actin, α-MHC, β-MHC, and TNC) in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–CACNB4 groups. C Fluo-3 staining showing intracellular Ca2+ levels in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–CACNB4 groups. D ELISA analysis of ATP content in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–CACNB4 groups (original magnification × 200 bars = 100 μm) **, p < 0.01; ***, p < 0.001. Results are from three repeated experiments

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  Fig. 4. Interaction between CACNB4 and RyR2 in the regulation of heart failure. A STRING analysis showing a potential interaction between CACNB4 and RyR2. B, C Western blot analysis of RyR2 protein expression in cardiomyocytes or heart tissue from sham, MI, and MI + OE–CACNB4 groups. D, E GST pull-down assay and co-immunoprecipitation (Co-IP) demonstrating an interaction between CACNB4 and RyR2, respectively. F Western blot analysis of RyR2 protein expression in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–RyR2 groups. G Western blot analysis of myocardial injury markers (TNI, α-actin, α-MHC, β-MHC, and TNC) in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–RyR2 groups. H ELISA analysis of ATP content in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–RyR2 groups. I Fluo-3 staining showing intracellular Ca2+ levels in cardiomyocytes from control, hypoxia, Hypoxia + OE–NC, and Hypoxia + OE–RyR2 groups (original magnification × 200 bars = 100 μm) *, p < 0.05; ***, p < 0.001. Results are from three or six repeated experiments

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  Fig. 5. Inhibition of RyR2 exacerbates heart failure in vivo. A Western blot analysis of RyR2 protein expression in heart tissue with TMDJ-035 treatment. B Echocardiography analysis of cardiac function indicators (LVIDd, LVIDs, EF%, FS%) in heart tissue with TMDJ-035 treatment. C Hematoxylin and eosin (H&E) staining of heart tissue in heart tissue with TMDJ-035 treatment (original magnification × 100 bars = 200 μm) (original magnification × 200 bars = 100 μm). D Masson staining showing collagen fiber deposition in heart tissue with TMDJ-035 treatment (original magnification × 100 bars = 200 μm) (original magnification × 200 bars = 100 μm). E Western blot analysis of myocardial injury markers (TNI, BNP, and TNC) in heart tissue with TMDJ-035 treatment. F, G ELISA analysis of ATP content and Ca2+ levels in heart tissue in heart tissue with TMDJ-035 treatment (original magnification × 200 bars = 100 μm) **, p < 0.01; ***, p < 0.001. Results are from six repeated experiments

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  Fig. 6. Inhibition of RyR2 exacerbates heart failure in vitro. A Western blot analysis of myocardial injury markers (TNI, α-actin, α-MHC, β-MHC, and TNC) in cardiomyocytes from Hypoxia + OE–CACNB4 + DMSO and Hypoxia + OE–CACNB4 + TMDJ-035 groups. B, C ELISA analysis of ATP content and Fluo-3 staining showing intracellular Ca2+ levels in cardiomyocytes from Hypoxia + OE–CACNB4 + DMSO and Hypoxia + OE–CACNB4 + TMDJ-035 group (original magnification × 200 bars = 100 μm) ***, p < 0.001. Results are from three repeated experiments