Retinol‐Binding Protein 4 Promotes Cardiac Injury After Myocardial Infarction Via Inducing Cardiomyocyte Pyroptosis Through an Interaction With NLRP3

Figures

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  Figure 1. RBP4 (retinol‐binding protein 4), expression is increased in acute myocardial infarction both in vivo and in vitro. A and B, RBP4 protein (A) and mRNA (B) levels in the border zone of infarct myocardium at day 1, day 3, and day 7 post‐acute myocardial infarction and sham controls; n=6; *P<0.05 vs sham; **P<0.01 vs sham. C, Immunofluorescence staining of heart tissue sections with anti‐RBP4 antibody (red), anti‐α‐actinin (green) and DAPI (blue). Scale bar=40 μm. For the construction of ischemia‐hypoxia model, primary cardiomyocytes were first serum starved overnight and then cultured with sugar‐ and serum‐free DMEM/F12 in hypoxic condition (1% O2) for 6 hours. D, Immunofluorescence staining of cardiomyocytes with anti‐RBP4 antibody (red), anti‐α‐actinin (green) and 4′,6‐diamidino‐2‐phenylindole (blue). Scale bar=80 μm. E and F, RBP4 protein (E) and mRNA (F) levels in cardiomyocytes treated with ischemia‐hypoxia; n=6; *P<0.05 vs control; **P<0.01 vs control. AMI indicates acute myocardial infarction; DAPI, 4′,6‐diamidino‐2‐phenylindole; I/H, ischemia/hypoxia; and RBP4, retinol‐binding protein 4.

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  Figure 2. RBP4 (retinol‐binding protein 4), regulates ischemia‐hypoxia‐induced oxidative stress and cell injury in cardiomyocytes. A, The indexes of oxidative stress including glutathione peroxidase, superoxide dismutase, malondialdehyde, and reactive oxygen species were measured at day‐3 post‐acute myocardial infarction and sham controls. B, Lactate dehydrogenase release in the myocardium at day‐3 post‐acute myocardial infarction and sham controls; n=6; *P<0.05 vs sham. C and D, Primary cardiomyocytes were treated with ischemia‐hypoxia for 6 hours. The indexes of oxidative stress including glutathione peroxidase, superoxide dismutase, malondialdehyde, and reactive oxygen species (C) as well as the cell viability (D) were measured; n=6; *P<0.05 vs control. E and F, Primary cardiomyocytes were transfected with adenovirus carrying shRBP4 or scramble RNA for 48 hours and then treated with ischemia‐hypoxia for 6 hours. The indexes of oxidative stress (E) and cell viability (F) were measured; n=6; *P<0.05 vs ischemia‐hypoxia+Ad scramble. G and H, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 or GFP for 48 hours. The indexes of oxidative stress…

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  Figure 3. RBP4 (retinol‐binding protein 4), expression is correlated with cardiac proptosis in acute myocardial infarction. A and B, The expression levels of NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3) inflammasome and pyroptosis‐related genes in the myocardium of acute myocardial infarction mice and sham controls were examined western blot (A) and quantitative real‐time polymerase chain reaction (B); n=6; *P<0.05 vs sham; **P<0.01 vs sham. C, Immunofluorescence staining of heart tissue sections with anti‐NLRP antibody (red) and 4′,6‐diamidino‐2‐phenylindole (blue) together with cell‐specific marker (green), including anti‐α‐actinin (cardiomyocyte), anti‐F4/80 (macrophage), and anti‐α‐SMA (fibroblast). Scale bar=40 μm. Arrows represent double positive cells. D and E, Serum interleukin‐1β (D) and interleukin‐18 (E) levels in acute myocardial infarction mice and sham controls. **P<0.01 vs sham; n=6 per group. F and G, Cardiac RBP4 protein concentrations are positively correlated with serum interleukin‐1β (F) and interleukin‐18 (G). H and I, Retinol and retinyl ester levels in the heart (H) and serum (I) in acute myocardial infarction mice…

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  Figure 4. RBP4 (retinol‐binding protein 4) regulates pyroptosis in cardiomyocytes. A and B, The protein (A) and mRNA (B) levels of NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3) inflammasome and pyroptosis‐related genes in cardiomyocytes treated with ischemia‐hypoxia for 6 hours; n=6; *P<0.05 vs ctrl; **P<0.01 vs ctrl. C, The percentage of propidium iodide (red) positive cells after ischemia‐hypoxia treatment for 6 hours. **P<0.01 vs ctrl. D, Lactate dehydrogenase release in cardiomyocytes treated with ischemia‐hypoxia for 6 hours. *P<0.05 vs ctrl. E through H, Primary cardiomyocytes were transfected with adenovirus carrying shRBP4 or scramble RNA for 48 hours and then treated with ischemia‐hypoxia for 6 hours. Protein levels of NLRP3 inflammasome and pyroptosis‐related genes were detected by western blot (F). The percentage of propidium iodide (red) positive cells (G) and Lactate dehydrogenase release (H) were reduced by knockdown of RBP4; n=6; *P<0.05 vs scramble; **P<0.01 vs scramble. I through L, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 or GFP for 48 hours. Protein levels of NLRP3…

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  Figure 5. RBP4 (retinol‐binding protein 4) regulates cardiomyocyte pyroptosis via interacting with NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3). A, Co‐immunoprecipitation identified a protein interaction between RBP4 and NLRP3 in primary cardiomyocytes. B through D, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 for 24 hours, and then transfected with NLRP3 siRNA or scramble for 24 hours. Protein levels of RBP4, NLRP3 inflammasome and pyroptosis‐related genes were detected by western blot (B). The percentage of propidium iodide (red) positive cells (C) and lactate dehydrogenase release (D) were reduced by silencing of NLRP3; n=6; *P<0.05 vs scramble; **P<0.01 vs scramble. E through G, Primary cardiomyocytes were transfected with adenovirus carrying the expression sequence of RBP4 for 24 hours, and then treated with NLRP3 inhibitor MCC950 (10 μmol/L) for 24 hours. Protein levels of RBP4, NLRP3 inflammasome and pyroptosis‐related genes were detected by western blot (E). The percentage of propidium iodide (red) positive cells (F) and lactate dehydrogenase release (G) were reduced by…

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  Figure 6. Knockdown of RBP4 (retinol‐binding protein 4) in heart attenuates cardiac pyroptosis and cardiac dysfunction in acute myocardial infarction. Adenovirus carrying shRBP4 was delivery to the myocardium by intrapericardial injection immediately after the ligation of left anterior descending. A, RBP4 mRNA level was detected in different tissues. B, Protein levels of RBP4, NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3) inflammasome, and pyroptosis‐related genes were detected by Western blot. C, Lactate dehydrogenase release in the left ventricular myocardium. D, Triphenyltetrazolium chloride staining of infarct size at day 3 after the ligation of left anterior descending. E, mRNA levels of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain 7 in the left ventricular myocardium. F, Echocardiography data including left ventricular ejection fraction, fractional shortening, left ventricular volume, and left ventricular internal dimension; n=5; *P<0.05 vs scramble; **P<0.01 vs scramble. AMI indicates acute myocardial infarction; ANP, atrial natriuretic peptide; ASC, apoptosis‐associated speck‐like protein containing…

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  Figure 7. Potential role of RBP4 (retinol‐binding protein 4) in acute myocardial infarction. In the process of acute myocardial infarction, RBP4 is upregulated in ischemic cardiomyocytes. RBP4 directly binds to and activates NLRP3 (nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3), which in turn induces Caspase‐1 mediated cardiomyocyte pyroptosis. AMI indicates acute myocardial infarction; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; GSDMD, gasdermin‐D; IL‐1β, interleukin‐1β; IL‐18, interleukin‐18; LDH, lactate dehydrogenase; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain‐containing 3; and RBP4, retinol‐binding protein 4.