Cardiac troponin I autoantibody induces myocardial dysfunction by PTEN signaling activation

Abstract

Background: The objective of the current study was to study the molecular mechanism(s) underlying cardiac troponin I autoantibody (cTnIAAb) binding to cardiomyocyte and resultant myocardial damage/dysfunction.

Methods: cTnIAAb was purified from serum of 10 acute myocardial infarction (AMI) patients with left ventricular remodeling. Recombinant human cTnI was used to generate three mouse-derived monoclonal anti-cTnI antibodies (cTnImAb1, cTnImAb2, and cTnImAb3). The target proteins in cardiac myocyte membrane bound to cTnImAb and effect of cTnIAAb and cTnImAb on apoptosis and myocardial function were determined.

Findings: We found that cTnIAAb/cTnImAb1 directly bound to the cardiomyocyte membraneα-Enolase (ENO1) and triggered cell apoptosis via increased expression of ENO1 and Bax, decreased expression of Bcl2, subsequently activating Caspase8, Caspase 3, phosphatase and tensin homolog (PTEN) while inhibiting Akt activity. This cTnIAAb-ENO1-PTEN-Akt signaling axis contributed to increased myocardial apoptosis, myocardial collagen deposition, and impaired systolic dysfunction.

Interpretation: Results obtained in this study indicate that cTnIAAb is involved in the process of ventricular remodeling after myocardial injury. FUND: The National Natural Science Foundation of China (Grant#: 81260026).

Keywords: Acute myocardial infarction; Cardiac troponin I autoantibody; Phosphatase and tensin homolog; Ventricular remodeling; α-Enolase.

Fig. 1

Characterization of purified human cTnIAAb. a. Purified human cTnIAAb was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining, which revealed two protein bands with sizes of ~51 kDa and ~28 kDa in line with the characteristic molecular weights of immunoglobulins. b. Human cTnIAAb bound to protein lysates purified from human atria (lane 1 from left), mouse ventricles (lane 2 from left), and recombinant full-length cTnI (lane 3 from left) at the position of ~24 kDa as revealed by Western blotting. Binding to protein lysates purified from mouse liver tissue was used as a negative control (lane 4 from left). Left panel, non-reduced protein sample, anti-cTnIAAb blot; middle panel, reduced protein sample, anti-cTnIAAb blot; right panel, reduced protein sample, anti-cTnImAb blot. c. Representative images of immunohistochemical staining showing positive staining of cTnIAAb in tissue sections prepared from human atria and mouse ventricles. Human IgG was used as a negative control. Bar = 50 μm. d&e. Representative images showing fluorescence immunostaining of primary cultured neonatal Sprague-Dawley rat cardiomyocyte using human cTnIAAb. d, bar = 50 μm. e, bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Human cTnIAAb binds membrane of cultured cardiomyocytes and causes cell apoptosis. a&b. Flow cytometric analysis results showing that cTnIAAb treatment for 48 h triggered apoptosis in neonatal rat cardiomyocytes. a, Annexin V-PI staining was used for flow cytometric analysis. Representative images are shown. b, Quantification of AnnexinV/PI staining from 3 independent experiments. *p = .000 (ANOVA analysis). c & d. Flow cytometric analysis results showing that cTnIAAb treatment for 48 h triggered apoptosis in neonatal rat cardiomyocytes. c, Active Caspase-3 staining was used for flow cytometric analysis. Representative images are shown. d, Quantification of Annexin-V/PI staining from 3 independent experiments. *p = .000 (ANOVA analysis). e. cTnIAAb treatment for 48 h significantly increased expression of Bax and decreased expression of Bcl2 as revealed by immune blot analysis. β-actin was used as a loading control. Shown are representative blots of 3 independent experiments. f. Quantification of immunoblot analysis using ImageJ software shown in e. n = 3, *p = .000 (ANOVA analysis). g. Human cTnIAAb specifically bound to the primary cultured myocardial cell membrane as revealed by cell membrane staining and flow cytometric analysis. Shown are representative images of 3 independent experiments.

Fig. 3

cTnImAbs as bioactive alternative for cTnIAAb. a. Differential binding affinities of cTnImAb1, −2, and −3 for myocardial membrane as revealed by flow cytometry. cTnImAb1 exhibited the highest binding force. b & c. Flow cytometry showed that cTnImAb1 was a potent competitor against human cTnIAAb in binding to the myocardial cell membrane. Shown are representative images (b) and quantification (c). *p = .000 (ANOVA analysis). d. Differential binding affinities of cTnImAb1, −2 and −3 for the membranes of BCG823 cells as revealed by flow cytometry. cTnImAb1 exhibited the highest affinity. e & f. Flow cytometry showed that cTnImAb1 was a potent competitor against human cTnIAAb in binding to the membrane of BCG823 cells. Shown are representative images (e) and quantification (f). *p = .000 (ANOVA analysis).

Fig. 4

cTnIAAb binds to α-enolase (ENO1) in myocardial cell membrane. a. The concentrated purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography was subjected to SDS-PAGE followed by Coomassie blue staining, which revealed one single band. b. The purified protein from BCG823 cell lysates specifically bound to ENO1 mAb, but could not bind to cTnImAb1 again. Left: Western blotting showing that the purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography could not interact with cTnImAb1 again. Middle: Western blotting showing that the purified protein from BCG823 cell lysates through cTnImAb1-dextran affinity chromatography could specifically bind to ENO1 mAb (51 KDa). Right: Western blotting showing that the non-reduced protein lysates from mouse cardiomyocytes interacted with cTnImAb1 at both 28 kDa and 51 kDa, while the reduced protein lysates interacted with cTnImAb1 only at 28 kDa. c. Immunoblot analysis showed that cTnImAb1 and anti-ENO1 pAb bound to the immunoprecipitation-purified endogenous dimeric ENO1 from cardiomyocytes. Far Left: Mouse cTnImAb1 strongly interacted with the unreduced ENO1 dimer (line 6, ~98 kDa) immunoprecipitated by rabbit ENO1 mAb155102, while it weakly interacted with the reduced endogenous ENO1 (line 3, ~47 kDa). Mouse cTnImAb1 interacted with myocardial cTnI (lines 2 and 5, ~28 kDa) as well as the unreduced input protein sample (line 2, ~98 kDa). Left: Mouse ENO1 pAb strongly interacted with the unreduced ENO1 dimer (line 6, ~98 kDa) immunoprecipitated by rabbit ENO1 mAb155102, while it weakly interacted with the reduced endogenous ENO1 (line 3, ~47 kDa). Mouse ENO1 pAb interacted with myocardial ENOI (lines 2 and 5, ~47 kDa) as well as the unreduced input protein sample (line 2, ~98 kDa). Right: Rabbit ENO1 mAb155955 interacted with ENO1 in the input protein sample (lines 2 and 5) as well as the immunoprecipitated endogenous ENO1 by rabbit ENO1 mAb155102 (lines 3 and 6). Rabbit ENO1 mAb155102 also bound to anti rabbit IgG (lines 4 and 7). Far Right: Mouse actin mAb interacted with the actin in the input protein sample (lines 2 and 5). All images are representative of at least 3 independent iterations. d. Identification of FLAG-tagged recombinant wild-type and C389A, C357A, C339A, C119A ENO1 protein expressed in 293 T cells. e. The reaction of recombinant ENO1 protein and 4 point-mutated ENO1 proteins with cTnImAb1 and Anti-ENO1 (non-reducing sample) respectively. Results shown are representative immunoblot analysis of three independent co-immunoprecipitation analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

cTnImAb1 upregulates the expression of ENO1 and induces cardiomyocyte apoptosis, while silencing ENO1 attenuates cTnImAb1–induced cardiomyocyte apoptosis. a & b. cTnImAb1 treatment (0, 25, 50, or 100 μg/ml) for 48 h induced cardiomyocyte apoptosis in a dose-dependent manner as revealed by flow cytometry. a, representative images. b, quantification of 3 independent experiments. *p = .000 (ANOVA analysis). c. cTnImAb1 treatment for 48 h at indicated doses increased Bax, cleaved Caspase3, and caspase8/p18 expression, and decreased Bcl2 expression in a dose-dependent manner as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images of 3 independent experiments. d&e. cTnImAb1 treatment (50 μg/ml) for indicated times induced cardiomyocyte apoptosis in a time-dependent manner as revealed by flow cytometry. d, representative images. e, quantification of 3 independent experiments, *p = .000 (ANOVA analysis). f. cTnImAb1 treatment (50 μg/ml) for indicated times increased Bax, cleaved Caspase3, and caspase8/p18 expression, and decreased Bcl2 expression in a dose-dependent manner as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images of 3 independent experiments. g-i. cTnImAb1 treatment (50 μg/ml) for 48 h increased ENO1 expression of primary cultured cardiomyocytes (i) compared with mouse IgG (g) and cTnTmAb (h) treatments as revealed by immunohistochemistry. Bar = 50 μm. j&k. cTnImAb1 treatment (50 μg/ml) for 48 h increased ENO1 expression compared with mouse IgG treatments as revealed by immunoblot analysis. β-actin served as a loading control. Shown are representative images (j) and quantification by ImageJ-mediated densitometry analysis (k) of 3 independent experiments. *p = .000 (t-test). l&m. Silencing ENO1 attenuated cTnImAb1 (50 μg/ml)-induced cardiomyocyte apoptosis compared with mock-siRNA group. l, Annexin V-PI staining was used for flow cytometric analysis. Representative images are shown. m, Quantification of Annexin-V/PI staining from 3 independent experiments. 24 h *p = .008, 48 h *p = .000 (t-test). n&o. RNAi-mediated silencing of Eno1 in primary cultured cardiomyocytes significantly reduced the cTnImAb1 (50 μg/ml)-induced cardiomyocyte apoptosis by increasing Bax expression and decreasing Bcl2 expression compared with mock-siRNA group. Representative immunoblot images are shown in n. ImageJ-based quantification of ENO1 (*ENO1-siRNA p = .000, Mock-siRNA p = .000), Bax (*ENO1-siRNA p = .000, Mock-siRNA p = .000), and Bcl2 (*ENO1-siRNA p = .003, Mock-siRNA p = .020) protein expression is shown in o. (ANOVA analysis).

Fig. 6

cTnImAb1 induces cardiac dysfunction and pathological changes in mouse heart. a & b. Representative images of echocardiography (a) and screen shots of catheterization (b) showing decreased cardiac function in mice of the cTnImAb1 group compared with the mouse IgG and cTnTmAb groups. c. Representative images of hematoxylin and eosin (H&E) staining showing epicardial calcified plaque formation in the mouse hearts of the cTnImAb1 group, which were not seen in those of the mouse IgG and cTnTmAb groups. Bar = 50 μm. d. Representative images of Masson's Trichrome staining showing interstitial collagen deposition in the mouse hearts of cTnImAb1 group, which were not seen in those of the mouse IgG and cTnTmAb groups. Bar = 50 μm. e & f. Representative images of TUNEL staining (e) and quantification (f) in the mouse hearts of the cTnImAb1 group compared with that in the mouse IgG and cTnTmAb groups. Bar = 10 μm. *p = .000 (ANOVA analysis).

Fig. 7

cTnImAb1 treatment increases ENO1 expression followed by activation of PTEN and suppression of Akt signaling in mouse hearts. a. Heat map from PathScan® Akt Signaling Antibody Array kit analysis showing changes in 16 phosphorylated proteins. b & c. Statistical analysis of heat map data in a and volcano plot showing that cTnImAb1 treatment significantly increased PTEN Ser380 phosphorylation in the mouse heart. 1. PTEN Ser380, *p = .023, (t-test). 2. GSK-3b Ser9, *p = .038, (t-test). 3. PTEN Ser380, *p = .031 (t-test). d&e. Immunoblot analysis showed that cTnImAb1 treatment significantly increased ENO1 expression and PTEN Ser380 phosphorylation but suppressed Akt Thr308 phosphorylation. β-actin served as a loading control. e is the statistical analysis of d. ENO1 *p = .002, pPTEN/PTEN/Actin, *p = .003, pAkt/Akt/Actin, *p = .003. (ANOVA analysis). f-h. cTnImAb1 treatment (50 μg/ml) induced changes in ENO1 expression and phosphorylation of PTEN Ser380 and Akt Thr308 at different time points in cultured cardiomyoyctes. cTnImAb1 increased the p-Ser380-PTEN/total PTEN ratio at 8 h but decreased the pThr308-AKT/total Akt ratio at 8–12 h. Mouse IgG treatment did not alter pSer380 PTEN but significantly increased the pThr308-Akt/total Akt ratio at 24–36 h. f shows representative blot images of three independent experiments. g and h represent ImageJ-based densitometry analysis of relative changes in p-Ser380-PTEN/total PTEN ratio (G, 8 h *p = .020, 12 h *p = .024, t-test) and pThr308-AKT/total Akt (H, 12 h *p = .018, 24 h *p = .001, 36 h *p = .001, t-test). i-l. Knockdown of ENO1 significantly suppressed cTnImAb1–induced alteration in PTEN/Akt signaling. Cultured cardiomyocytes were transfected with siRNA targeting Eno1 and then subjected to cTnImAb1 treatment (50 μg/ml) for 4–48 h as indicated. Mock-siRNA was used as a control. Depletion of Eno1 suppressed the p-PTEN Ser380/total PTEN ratio and increased the pAkt-Thr308/total Akt ratio at 4, 8, and 12 h, respectively. i shows representative blot images of three independent experiments. j represents ImageJ-based densitometry analysis of relative changes in ENO1 protein levels (J,48 h *p = .000, t-test). k and l represent ImageJ-based densitometry analysis of relative changes in p-Ser380-PTEN/total PTEN ratio (k, 48 h *p = .000, t-test) and pThr308-AKT/total Akt (l, 6 h *p = .000, 12 h *p = .000, t-test).

Fig. 8

Graphical mechanism summary: Human cTnIAAb interacts with ENO1 present in the myocardial membrane and induces apoptosis of cultured cardiomyocytes. Human cTnIAAb can trigger cardiomyocyte apoptosis via binding membrane ENO1, increasing ENO1 expression, promoting phosphorylation of PTEN Ser380, and suppressing Akt activity in the mouse model of cTnImAb-induced myocardial injury. This induces the pro-apoptotic Bax and suppresses the anti-apoptotic Bcl2 expression resulting in cardiomyocyte apoptosis.

Supplementary Fig. 1

Effect of cTnIAAb treatment on the expression of hypertrophic genes in vitro. Neonatal rat cardiomyocytes were treated for 48h with cTnIAAb (100 μg/L), cTnTmAb (100 μg/L), mouse IgG (100μg/L) and AngII (1μM, Sigma), respectively. AngII was used as positive control. Thereafter, total RNA was isolated using TRIzol reagent, and the hypertrophic markers, Nppa, Nppb and myh7/myh6 were quantified using RT-PCR and normalized to β-actin housekeeping gene. *p<0.05 compared to control (t-test, n=3).

Supplementary Fig. 2

Representative immunofluorescence images showing co-localization of cTnIAAb and cell membrane marker as revealed by immunofluorescence confocal microscopy. Green, cTnlAAb; red, Dil, a membrane marker; blue, nuclei. Bar = 10 μm.

Supplementary Fig. 3

Mass spectrometric analysis and Mascot search results determined that ENO1 was the protein purified from BCG823 cell lysates through cTnImAb1–dextran affinity chromatography. A. Mass spectrum from mass spectrometric analysis. B. Secondary mass spectrum. C. Mascot search results showed that the only protein with a statistical significance was ENO1.

Supplementary Fig. 4

cTnTmAb and mouse-IgG did not induce apoptosis in primary cultured cardiomyocytes. A. Treatment with mouse-IgG at different concentrations (0, 25μg/ml, 50μg/ml, 100μg/ml) or 50μg/ml mouse-IgG for 48h did not induce myocardial apoptosis as revealed by FACS. B. Treatment with cTnTmAb at different concentrations (0, 25μg/ml, 50μg/ml, 100μg/ml) or 50μg/ml cTnTmAb for 48h did not induce myocardial apoptosis as revealed by FACS. C. Treatment with mouse-IgG at different concentrations (0, 25μg/ml, 50μg/ml, 100μg/ml) or 50μg/ml mouse-IgG for 48h did not alter the expression of Bax, Bcl2 and cleaved Caspase3 as revealed by immunoblot analysis. β-actin served as a loading control. D. Treatment with cTnTmAb at different concentrations (0, 25μg/ml, 50μg/ml, 100μg/ml) or 50μg/ml cTnTmAb for 48h did not alter the expression of Bax, Bcl2 and cleaved Caspase3 as revealed by immunoblot analysis. β-actin served as a loading control.

Supplementary Fig. 5

cTnImAb1 significantly up-regulated Eno1 mRNA expression in primary cultured cardiomyocytes compared with mouse-IgG and cTnTmAb groups.

Supplementary Fig. 6

Effects of cTnImAb1, cTnTmAb, and mouse-IgG on the expression of mRNAs encoding matrix metalloproteinase and matrix metalloproteinase inhibitors in mouse hearts. cTnImAb1 treatment significantly increased the expression of matrix metalloproteinase-3 (Mmp3) but not Mmp12 and metal matrix metalloproteinase inhibitor of matrix protein 3 (Timp3) in the hearts compared with treatment with either cTnTmAb or mouse IgG. *p<0.05 vs. cTnTmAb and mouse IgG(t-test, n=3).

Supplementary Fig. 7

Representative global view of the harvested hearts comparing the cTnImAb1, cTnTmAb and mouse IgG treatment groups (Group cTnImAb1 n=21, Group cTnTmAb n=13, Group mouse IgG n=14).

Supplementary Fig. 8

cTnImAb1 treatment did not increase inflammatory cell infiltration in the mouse heart compared with the cTnTmAb and mouse IgG groups. Immunohistochemical staining against CD4, CD8, CD20, and CD68 was performed on heart tissue sections. Representative images are shown (Group cTnImAb1 n=21, Group cTnTmAb n=13, Group mouse IgG n=14).