Low-Density Lipoprotein Receptor–Related Protein-1 Is a Therapeutic Target in Acute Myocardial Infarction

Figure 1. SP16 Is an LRP1 Agonist When serine protease inhibitors (SERPINs) bind the preoteases inactivating them, a conformational change occurs by which a short peptide containing a unique motif (5 to 11 amino acids) is exposed. SP16 contains alpha-1 antitrypsins (AATs) pentapeptide FVFLM responsible for binding to low-density lipoprotein receptor–related protein-1(LRP1). (A) Scheme modified from Joslin et al. (13) and Lillis et al. (3). (B) SP16 binds LRP1 in vitro, whereas SP34 does not. (C,D) SP16 inhibits NF-κB signaling induced by lipopolysaccharide (LPS) or Gp96 in THP1-XBlue-MD2-CD14 cells, and that treatment with LRP1 blocking antibody limits SP16-related inhibition. Ab = antibody; IgG = immunoglobulin G.

Figure 2. LRP1 Expression Is Increased in Heart During Acute Myocardial Infarction (A) Low-density lipoprotein receptor–related protein-1 (LRP1) expression is significantly increased in the heart of patients with end-stage heart failure due to prior myocardial infarction undergoing heart transplantation, compared with subjects who died of noncardiac causes (p < 0.001; n = 6 per group). (B) Increased LRP1 expression is seen in the heart of mice subject to myocardial ischemia (30 min) and reperfusion (24 h) (top right panel) (p = 0.003, n = 4 per group). The immunofluorescence for LRP1 (red) and cardiac muscle actin (green) showing (C) increased LRP1 expression in areas bordering the infarct zone, (D) compared with sham-operated mice. GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. SP16 Induces Rapid Phosphorylation of Akt During Ischemia–Reperfusion We measured phosphorylation at Ser473 of Akt in myocardial samples during ischemia–reperfusion as a measure of the prosurvival effect of SP16. As compared with vehicle-treated mice, mice receiving SP16 100 μg intraperitoneally at time of reperfusion had a significant increase in pAkt/total Akt ratio, associated with a reduction in the ratio of the proapoptotic to antiapoptotic Bax/Bcl2 ratio and of caspase-3 myocardial tissue activity. i.p. = intraperitoneal; p-Akt = phosphorylated Akt.

Figure 4. Infarct-Sparing Effect of SP16 We induced acute myocardial infarction (AMI) in adult male mice by coronary ligation and transient ischemia (30 min) followed by reperfusion. When compared with vehicle solution, SP16, but not SP34, given intraperitoneally at reperfusion significantly reduced in a dose-dependent fashion infarct size, measured as percent of area at risk at tetrazolium chloride (TTC) staining at pathology and preserved left ventricular systolic function measured as left ventricular (LV) fractional shortening (LVFS) %. n = 5 to 8 mice/group. c-Trop I = cardiac troponin I; LVEDD = left ventricular end-diastolic diameter; LVESD = left ventricular end-systolic diameter.

Figure 5. Effects of SP16 on LV Systolic Function We measured left ventricular (LV) systolic function with SP16 at rest and after challenge with isoproterenol to evaluate also contractile reserve. SP16 had no significant effect on LV fractional shortening (LVFS) (A) at rest or (B) after isoproterenol challenge. These data show that, as tested, SP16 has no appreciable inotropic activity in vivo. We also measured LVFS at 7 days after acute myocardial infarction (AMI) in mice. (C) Mice treated with SP16 at reperfusion had significantly greater LVFS than did vehicle-treated mice. (D) This was associated with a significantly smaller area of wall motion abnormality, a surrogate for infarct size. These data show that the early cardioprotective effects of SP16 are maintained at 7 days after surgery. n = 5 to 8 mice/group. d = days.

Figure 6. Effects of SP16 on Cardiomyocyte Apoptosis and Myocardial Inflammation and Fibrosis We measured the percentage of (A) apoptotic cardiomyocytes by detecting nuclei showing in situ end labeling of deoxyribonucleic acid fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) in the peri-infarct border zone, (B) the number of leukocytes (positive for cluster of differentiation 45) per high-power field (HPF) ×40, and (C) the extent of the fibrotic infarct scar (at Masson trichrome stain) in the mouse hearts 7 days after 30 min of ischemia. Treatment with SP16 led to a significant reduction in infarct scar size and a reduction in cardiomyocyte apoptosis and myocardial inflammation in the peri-infarct border zone. n = 4 mice/group. LV = left ventricle.

Figure 7. LRP1 Mediates SP16-Induced Cardioprotection We pretreated mice with a Low-density lipoprotein receptor–related protein-1 (LRP1) blocking antibody to test whether LRP1 mediated the cardioprotective signal of SP16 and alpha-1 antitrypsins (AAT). Treatment with the blocking antibody eliminated the protective effects of both SP16 and plasma-derived AAT. n = 5 to 8 mice/group. Ab = antibody; LV = left ventricular.