PCSK9 inhibition alleviates sepsis-induced myocardial dysfunction by facilitating PINK1/parkin-associated mitophagy

Figures

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  FIGURE 1. PCSK9 expression in hearts and serum are increased in cecal ligation and puncture (CLP) mice. (A) Representative immunofluorescent images co-stained with PCSK9 and WGA of heart tissue from CLP mice (scale bar = 50 μm, magnification: ×20). (B) Statistical assessment of PCSK9 relative fluorescent intensity. (C) Representative Western blot images of PCSK9 in heart tissue of mice with CLP. (D) Quantitative analysis of PCSK9 expression in Western blot images. (E) Quantification of serum PCSK9 levels in CLP mice by colorimetry. N = 6 per group. **, p < 0.01 versus Sham group.

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  FIGURE 2. PCSK9 inhibitor alleviates CLP-induced heart dysfunction in mice. (A) Experimental timeline and workflow diagram (created with BioRender.com) (B) 7-day survival rates of mice post-CLP across groups (n = 20 per group). (C) Representative echocardiographic snapshots of the left ventricle. (D) Quantification of left ventricular internal diameter in diastole (LVIDd) (E) left ventricular internal diameter in systole (LVIDs) (F) left ventricular ejection fraction (LVEF) and (G) left ventricular fraction shortening (LVFS) from ultrasound images. (H) Typical right ventricular systolic pressure (RVSP) curve. (I) Quantification of RVSP. n = 6 per group. *, p < 0.05 and **, p < 0.01 versus Sham group. #, p < 0.05 and ##, p < 0.01 versus CLP group.

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  FIGURE 3. PCSK9 inhibitor ameliorates cardiac injury, apoptosis and inflammation in mice with sepsis-induced myocardial dysfunction (SIMD). (A) Representative histopathological structure (scale bar = 500 μm, magnification: ×2, zoomed scale bar = 20 μm, zoomed magnification: ×40) and TUNEL immunohistochemical images (scale bar = 20 μm, magnification: ×40) of mice hearts. (B) Quantitative analysis of positive rate of TUNEL immunohistochemistry. (C) Serum concentration of cardiac troponin I (cTnI) (D) creatine kinase muscle-brain (CK-MB) (E) tumor necrosis factor-α (TNF-α) (F) interleukin-1β (IL-1β) and (G) interleukin-6 (IL-6) were detected via colorimetric assay. N = 6 per group. *, p < 0.05 and **, p < 0.01 versus Sham group. #, p < 0.05 and ##, p < 0.01 versus CLP group.

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  FIGURE 4. PCSK9 inhibitor attenuates oxidative stress and the imbalance between mitochondrial damage and mitochondrial dynamics in SIMD mice heart. (A) Quantification of superoxide dismutase (SOD) (B) glutathione (GSH) (C) malondialdehyde (MDA) (D) reactive oxygen species (ROS) and (E) adenosine triphosphate (ATP) in heart tissue were measured using colorimetric analysis. (F) Representative snapshots of cardiac TEM (scale bar = 1 μm, magnification: 10000×), yellow arrows indicate normal mitochondria, red arrows denote abnormal mitochondria (e.g., swollen with disrupted cristae), and green arrows represent mitochondrial dynamics (fission or fusion). (G) Illustrative Western blot images of Drp1 and Mfn2 in heart. (H) Statistical analysis of Drp1 and (I) Mfn2 of Western blot images. N = 6 per group. *, p < 0.05 and **, p < 0.01 versus Sham group. #, p < 0.05 and ##, p < 0.01 versus CLP group.

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  FIGURE 5. PCSK9 inhibitor improves mitophagy via PINK1/Parkin pathway in heart of mice with SIMD. (A) Representative images of cardiac TEM (scale bar = 1 μm, magnification: 10000×), yellow arrows indicate normal mitochondria, red arrows denote abnormal mitochondria (e.g., swollen with disrupted cristae), and blue arrows represent autophagosomes (AP) or autolysosomes (AL). (B) Illustrative Western blot images of key biomarkers during mitophagy process. (C) Quantification of PCSK9 (D) SQSTM1 (E) LC3 (F) PINK1 (G) Parkin (H) TOM20 and (I) TIM23 in Western blot images. (J) Statistical assessment of mtDNA to nDNA ratio and (K) the copies number of mtDNA using qPCR assay. N = 6 per group. *, p < 0.05 and **, p < 0.01 versus Sham group. #, p < 0.05 and ##, p < 0.01 versus CLP group.

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  FIGURE 6. Inhibition of mitophagy aggregates mitochondrial damage and oxidative stress in mice with SIMD after PCSK9 blockage. (A) Representative cardiac TEM snapshots (scale bar = 1 μm, magnification: 10000×). Yellow arrows indicate normal mitochondria, red arrows denote abnormal mitochondria (e.g., swollen with disrupted cristae), green arrows highlight mitochondrial dynamics (fission or fusion) and blue arrows represent autophagosomes (AP) or autolysosomes (AL). (B) Illustrative Western blot images towards typical biomarkers of mitophagy. (C) Quantification of PCSK9 (D) Drp1 (E) Mfn2 (F) SQSTM1 (G) LC3 (H) PINK1 (I) Parkin (J) TOM20 and (K) TIM23 in Western blot images (L) mtDNA to nDNA ratio and (M) copies number of mtDNA were measured via qPCR (N) ATP (O) ROS (P) SOD and (Q) MDA concentrations in hearts were detected using colorimetric analysis. N = 6 per group. Ns, not significant. *, p < 0.05 and **, p < 0.01 versus Sham group. #, p < 0.05 and ##, p < 0.01 versus CLP group. and, p < 0.05 and andand, p < 0.01 versus EVO group.

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  FIGURE 7. Inhibition of mitophagy deteriorates cardiac dysfunction, injury, inflammation and apoptosis in mice with SIMD following PCSK9 blockage. (A) Representative echocardiography snapshots towards left ventricle. (B) Quantification of LVEF in echocardiography snapshots. (C) Typical RVSP curve across different groups. (D) Statistical analysis of RVSP (E) Serum cTnI (F) TNF-α and (G) IL-1β concentrations were measured by colorimetry assay. (H) Illustrative histopathological and TUNEL immunohistochemical images of heart tissue (scale bar = 20 μm, magnification: ×40). (I) Quantitative assessment to apoptosis positive rate. N = 6 per group. **, p < 0.01 versus Sham group. ##, p < 0.01 versus CLP group. andand, p < 0.01 versus EVO group.

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  FIGURE 8. Mitophagy inhibitor exacerbates cellular injury, inflammation, oxidative stress and mitochondrial damage via restraining mitochondrial dynamics in HL-1 cells. (A) Viability of HL-1 cells with varying PCSK9 inhibitor concentrations and (B) over a time gradient were measured by CCK8 assay. The dashed boxes indicate the EVO concentrations and intervention times selected for subsequent cellular experiments. (C) Concentration of cTnI (D) TNF-α (E) IL-1β (F) SOD and (G) MDA in supernatant were captured using colorimetric assay. (H) Quantitative analysis of ATP level in cells. (I) Representative fluorescent images of cleaved-caspase 3 (scale bar = 20 μm, magnification: ×40) and ROS (scale bar = 100 μm, magnification: ×20) in HL-1 cells. (J) Illustrative flow cytometry plots of ROS in HL-1 cells. (K) Typical Western blot snapshots of Drp1 and Mfn2. (L) Quantitative analysis of Drp1 and (M) Mfn2 expression level in Western blot images. N = 3 per group. Ns, not significant. *, p < 0.05; **, p < 0.01 and ***, p < 0.001.

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  FIGURE 9. Mdivi-1 reverses enhancement of PINK1/Parkin-induced mitophagy in HL-1 cells following subsequent to PSCK9 inhibition. (A) Representative fluorescent images (scale bar = 50 μm, magnification: ×20) and flow cytometry plots of JC-1 in cells. (B) Quantification of JC-1 relative fluorescent intensity in fluorescent images. (C) Quantification of JC-1 mean fluorescent intensity in flow cytometry plots. (D) Typical TEM snapshots of HL-1 cells (scale bar = 500 nm, magnification: 20000×). Yellow arrows indicate normal mitochondria, red arrows denote abnormal mitochondria (e.g., swollen with disrupted cristae), green arrows highlight mitochondrial dynamics (fission or fusion) and blue arrows represent autophagosomes (AP), autolysosomes (AL) or phagophores (PP). (E) Illustrative immunofluorescent confocal images of TOM20 and LAMP1 in cells (scale bar = 50 μm, magnification: 60×, zoomed scale bar = 10 μm, zoomed maginification: 120×). (F) Representative Western blot images of key mitophagy biomarkers in cells. (G) Quantitative analysis of PCSK9 (H) SQSTM1 (I) LC3 (J) PINK1 (K) Parkin (L) TOM20 and (M) TIM23 expression level in Western blot images (N) mtDNA to nDNA ratio and (O) copies number…