Anti‐signal recognition particle antibodies induce cardiac diastolic dysfunction via oxidative stress injury

Figures

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  Figure 1. Immunoreactivity study of human source anti‐SRP antibodies. (a) Western blot of recombinant SRP54 protein revealed by mouse anti‐SRP antibody (line on the left) or immunoaffinity‐purified human anti‐SRP‐specific IgG (line on the right). (b–i) Representative immunofluorescence staining of mouse myocardium after incubation with (b) mouse anti‐SRP antibody and FITC‐labelled anti‐mouse secondary antibody, (c) immunoaffinity‐purified anti‐SRP‐specific IgG from patient plasma and FITC‐labelled anti‐human secondary antibody, (d) purified total IgG from HCs' plasma and FITC‐labelled anti‐human secondary antibody, (e) immunoaffinity‐purified anti‐SRP‐specific IgG from patient plasma with prior inhibition by free SRP54 protein and FITC‐labelled anti‐human secondary antibody and (f–i) immunoaffinity‐purified anti‐SRP‐specific IgG from patient plasma (green) and co‐staining with WGA (red) (Scale bar, 50 μm; 20 μm for inset). All are counterstained with DAPI (blue). These experiments were repeated three times. SRP, signal recognition particle; WGA, wheat germ agglutinin.

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  Figure 2. In vivo study of the pathogenicity of human source anti‐SRP antibodies. (a) Experimental overview. (b, c) Body weight (b) and wire hanging time (c) of mice before and after injection of total IgG (n = 8). (d) H&E staining of muscle cryosection in each group (Scale bar, 50 μm). (e) Quantification of necrotic muscle fibres in triceps muscles in each group (n = 5 per group from 5 random fields per mice). (f) Immunohistological staining of F4/80+ macrophages in mice muscle fibres in each group (Scale bar, 50 μm). (g) The relative value of F4/80 positive levels in each group (n = 5 per group from 5 random fields per mouse). (h–k) The comparison of serum levels of CK (h), CK‐MB (i), AST (j) and LDH (k) between two mice groups. (l–o) The comparison of serum levels of BNP (l), cTnT (m), cTnI (n) and LDH1 (o) between two mice groups (n = 8). All data are presented as mean ± SEM. The data shown are from one of three experiments, which all showed similar results. ns P > 0.05, *P < 0.05, **P < 0.01. AST, aspartate aminotransferase; BNP, B‐type natriuretic peptide; CK, creatine kinase; CK‐MB, creatine kinase‐MB; cTnI, cardiac troponin I; cTnT, cardiac troponin T; CTX, cyclophosphamide; H&E,…

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  Figure 3. The decline in cardiac diastolic function in mice after injection of human source anti‐SRP antibodies. (a) Representative images of left ventricular M‐mode echocardiographic tracings of mice after injection with HC or anti‐SRP IgG. (b, c) Percentages of LVEF (b) and LVFS (c) in two mice groups (n = 8). (d) Representative mitral flow patterns from pulsed‐wave colour Doppler echocardiography in two mice groups. (e, f) The levels of E/A ratio (e) and deceleration time (f) in two mice groups (n = 8). (g) Representative images of mitral annular Doppler tissue imaging in two mice groups. (h, i) The levels of IVS E/E′ ratio (h) and LW E/E′ ratio (i) in two mice groups (n = 8). (j–l) Representative steady‐state left ventricular pressure–volume (P–V) loops in two mice groups. (m–p) The diastolic stiffness coefficient β of EDPVR (m), the ESPVR (n), systolic contractility (o) and diastolic relaxation (p) were calculated from the P–V loop analysis in two mice groups (n = 5). All data are presented as mean ± SEM. The data shown are from one of three experiments, which all showed similar results. ns P > 0.05, *P < 0.05, **P < 0.01. HC, health control; IgG, immunoglobulin G; SRP, signal…

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  Figure 4. Cardiac serological and histological changes in mice after injection of human source anti‐SRP antibodies. (a) H&E staining (upper) and immunohistological staining for F4/80+ macrophages (lower) of myocardium in each group (Scale bar, 50 μm). (b) The relative value of F4/80 positive levels in each group (n = 5 per group from 5 random fields per mouse). (c) Representative immunofluorescent image of WGA staining of mice hearts in each group (Scale bar, 50 μm; green, WGA; blue, DAPI). (d) The relative value of myocardial cell size of mice in each group (n = 5 per group from 5 random fields per mouse). (e) Sirius red staining of myocardium in each group (Scale bar, 1 mm; 50 μm for inset). (f) Percentage of cardiac fibrosis area in each group (n = 5 per group from 5 random fields per mouse). (g) Representative immunofluorescent image of TUNEL staining of mice hearts in each group (Scale bar, 50 μm; green, TUNEL; blue, DAPI). (h) The relative value of TUNEL positive levels in each group (n = 5 per group from 5 random fields per mice). All data are presented as mean ± SEM. The data shown are from one of three experiments, which all showed similar results. ns P > 0.05. H&E,…

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  Figure 5. The upregulation of reactive oxygen species metabolic pathways in the transcriptomic analysis of mouse hearts. (a) Volcano plot showing significantly decreased (blue) and increased (red) genes. (b) Gene ontology (GO) analysis of RNA‐sequencing data. (c, d) Gene set enrichment analysis of ‘Positive Regulation of Reactive Oxygen Species Metabolic Process’ (c) and ‘Mitochondrial Membrane Organization’ (d) pathways with all detected mRNAs (n = 3 per group).

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  Figure 6. The alteration of mitochondrial integrity and function induced by increased ROS levels in mouse hearts. (a, b) Representative image (a) and summary fluorescence intensity (b) of DHE staining of myocardium for ROS formation (n = 5 per group from 5 random fields per mice; Scale bar, 50 μm). (c) Representative transmission electron microscopy images of cardiac tissue in each group (Scale bar, 5 μm; 1 μm for inset). (d–g) Quantification analysis of mitochondrial area (d), mitochondrial perimeter (e), mitochondrial form factor (f) and mitochondrial circularity (g) in each group (330 mitochondria per group). (h) Real‐time mitochondrial respiration capacity monitoring the oxygen consumption rate in primary cardiomyocytes isolated in each group. (i–k) The basal respiration (i), maximal respiration (j) and spare respiratory capacity (k) of primary cardiomyocytes isolated in each group (n = 5). All data are presented as mean ± SEM. The data shown are from one of three experiments, which all showed similar results. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. ROS, reactive oxygen species; IgG, immunoglobulin G; SRP, signal recognition particle; FCCP, carbonyl cyanide‐4…

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  Figure 7. NAC reversed the oxidative stress effect of immunoaffinity‐purified‐specific anti‐SRP antibodies on neonatal rat ventricular myocytes. (a, b) Representative image (a) and summary fluorescence intensity (b) of DHE staining of neonatal rat ventricular myocytes cultured with immunoaffinity‐purified‐specific anti‐SRP IgG or purified IgG from HCs, treated with or without NAC (n = 3 per group from 3 random fields; Scale bar, 50 μm). (c) Real‐time mitochondrial respiration capacity monitoring the oxygen consumption rate in neonatal rat ventricular myocytes in each group. (d, e) The maximal respiration (d) and proton leak (e) in neonatal rat ventricular myocytes in each group (n = 5). All data are presented as mean ± SEM. The data shown are from one of three experiments, which all showed similar results. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. DHE, dihydroethidium; FCCP, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone; IgG, immunoglobulin G; NAC, N‐acetylcysteine; OCR, oxygen consumption rate; SRP, signal recognition particle.