Fig 1: Knockin mice expressing phospho-mimetic Phb2S91 are less vulnerable to SC. Heterozygous Phb2S91D/+ mice, homozygous Phb2S91D/D mice, and wild-type (WT) mice aged 8–10 weeks were injected intraperitoneally with 10 mg/kg LPS to induce SC. Mice administered an equal volume of phosphate buffer saline served as controls. (A, B) Western blot analysis of cardiac p-PHB2S91 in WT, heterozygous Phb2S91D/+, and homozygous Phb2S91D/D mice treated with PBS or LPS. Total-PHB2 was used as the loading control. (C, D) Western blot analysis of total-PHB2 in WT, heterozygous Phb2S91D/+, and homozygous Phb2S91D/D mice treated with PBS or LPS. α-tubulin was used as the loading control. (E) The CO1 gene of mtDNA and the NDUFV1 gene of nDNA were amplified using qPCR to assess the relative ratio of mtDNA/nDNA in heart tissues obtained from WT, heterozygous Phb2S91D/+, and homozygous Phb2S91D/D mice under normal physiological conditions. (F-L) Echocardiographic evaluation. LVDd, left ventricular diastolic dimension; LVDs, left ventricular systolic dimension; IVS, interventricular septum thickness; E/A, ratio of early to late transmitral flow velocities; FS, ratio of left ventricular fractional shortening. (M-O) ELISA-based analysis of serum TnI, CK-MB, and LDH concentrations. (P-R) Transcriptional analysis of cardiac Mmp9, Mcp1, and Tnfα expression by qPCR. (S) Representative images of TnT and Gr-1 immunohistochemistry in cardiac sections. (T) Quantification of Gr-1-positive neutrophils in mouse heart tissues. (U) ELISA-based measurement of caspase-3 activity in heart tissues. Values are presented as mean ± SEM. For in vivo data, n = 6 mice per group. For in vitro data, n = 4 independent experiments. #p < 0.05, and ##p < 0.01
Fig 2: PKM2 induces PHB2 phosphorylation. PKM2 transgenic (PKM2Tg) mice and wild-type (WT) mice aged 8–10 weeks were injected intraperitoneally with 10 mg/kg LPS to induce SC. In vivo measurements were performed after 24 h later, and mice administered an equal volume of phosphate buffer saline served as controls. Immortalized mouse cardiac muscle HL-1 cells were treated with 10 µg/mL of LPS for 24 h to simulate SC in vitro. Cells treated with an equal volume of phosphate buffer saline were used as controls. Before LPS treatment, cardiomyocytes were transduced with Adenovirus encoding PKM2 (Ad-PKM2). β-gal-overexpressing (Ad-β-gal) cells were used as controls. (A) Mapping of PHB2 regions and deletion mutants. (B) Representative immunoblot from immunoprecipitation analysis of HL-1 cells transfected with PHB2 deletion mutants. (C-F) Co-IP analysis of the interaction between dimeric and tetrameric PKM2 forms and PHB2 in HL-1 cells. (G-I) Western blot analysis of p-PHB2S176 and p-PHB2Ser91 expression in cardiac tissues. (J, K) Representative results of an in vitro kinase assay evaluating binding of phospho-PHB2 variants to PKM2 in the presence of LPS or compound 3k, a PKM2 inhibitor. (L, M) Pulse-chase analysis of PHB2 protein half-life in HL-1 cells transfected with PHB2S91D and PHB2S91A mutant constructs. Values are presented as mean ± SEM. For in vivo data, n = 6 mice per group. For in vitro data, n = 4 independent experiments. #p < 0.05, and ##p < 0.01
Fig 3: PKM2 binds to and prevents PHB2 degradation. PKM2 transgenic (PKM2Tg) mice and wild-type (WT) mice aged 8–10 weeks were injected intraperitoneally with 10 mg/kg LPS to induce SC. In vivo measurements were performed after 24 h later, and mice administered an equal volume of phosphate buffer saline served as controls. Immortalized mouse cardiac muscle HL-1 cells were treated with 10 µg/mL of LPS for 24 h to simulate SC in vitro. Cells treated with an equal volume of phosphate buffer saline were used as controls. Before LPS treatment, cardiomyocytes were transduced with Adenovirus encoding PKM2 (Ad-PKM2). β-gal-overexpressing (Ad-β-gal) cells were used as controls. (A) Analysis of the effect of LPS exposure on cardiac Phb2 mRNA expression levels in WT and PKM2Tg mice. (B, C) Western blots analysis of cardiac PHB2 protein levels in WT and PKM2Tg mice. (D, E) Analysis of the half-life of PHB2 protein in HL-1 cells (pulse-chase assay). (F-G) Western blot analysis of PHB2 expression in HL-1 cells exposed to LPS in the presence or absence of MG132, a proteasome inhibitor, or betulinic acid (BA), a proteasome activator. (H, I) Pulse-chase analysis of PHB2 degradation rate in cultured in HL-1 cells treated with LPS and MG132. (J, K) Pulse-chase analysis of PHB2 degradation rate in cultured in HL-1 cells treated with LPS and BA. (L-M) Mapping of PHB2/PKM2 interacting regions by docking analysis. (N, O) Co-IP analysis of PKM2/PHB2 binding in HL-1 cells transfected with different domain deletion PKM2 mutants. (P) Mapping of PKM2 regions and deletion mutants. (Q) Western blot analysis of PHB2 expression in HL-1 cells transfected with domain deletion PKM2 mutants. Values are presented as mean ± SEM. For in vivo data, n = 6 mice per group. For in vitro data, n = 4 independent experiments. #p < 0.05
Fig 4: PHB2 dephosphorylation abolishes PKM2-mediated mitochondrial protection. HL-1 cardiomyocytes were transfected with PKM2 overexpression adenovirus (Ad-PKM2), a phosphorylation-defective PHB2S91A mutant, or a phosphorylation-mimetic PHB2 S91D mutant before LPS treatment. Adenovirus loaded β-gal (Ad-β-gal) cells were used as controls. (A-C) Immunofluorescence analysis of mitochondrial morphology in HL-1 cells. Representative images of mitochondria immunofluorescence (A), average mitochondrial length (B), and proportion of cardiomyocytes with fragmented mitochondria (C) are shown. (D-J) Western blot analysis of Drp1, Fis1, Mfn2, Opa1, Parkin, and Atg5 in HL-1 cells. (K, L) Mitophagy analysis results (mt-Keima assay). (M-P). Transcriptional analysis of Tfam, Nrf2, mtHsp70, and Atf6 expression by qPCR. Values are presented as mean ± SEM. For in vivo data, n = 6 mice per group. For in vitro data, n = 4 independent experiments. #p < 0.05, and ##p < 0.01
Fig 5: PKM2-mediated cardiomyocyte protection against septic insult requires PHB2 phosphorylation. HL-1 cardiomyocytes were transfected with PKM2 overexpression Adenovirus (Ad-PKM2), a phosphorylation-defective PHB2S91A mutant, or a phosphorylation-mimetic PHB2 S91D mutant before LPS treatment. Adenovirus loaded β-gal (Ad-β-gal) cells were used as controls. (A, C) ELISA-based analysis of TnI, CK-MB, and LDH levels in culture media of HL-1 cells transfected with phosphorylation-defective (PHB2S91A) and phosphorylation-mimetic (PHB2S91D) mutant constructs. (D) Cell viability analysis via CCK-8 assay in vitro. (E, F) Apoptosis analysis by TUNEL staining in cultured HL-1 cells. (G, H) Transcriptional analysis of Tnfα and Mcp1 expression. (I, J) Representative images of myosin immunofluorescence. Myosin expression levels were normalized to those of the control group. Values are presented as mean ± SEM. For in vivo data, n = 6 mice per group. For in vitro data, n = 4 independent experiments. #p < 0.05, and ##p < 0.01
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