P2Y12 Inhibition in Murine Myocarditis Results in Reduced Platelet Infiltration and Preserved Ejection Fraction

Abstract

Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y₁₂ receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the "ligand-induced binding sites" of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y₁₂ inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.

Keywords: P2Y12 inhibition; activated platelets; echocardiography; molecular imaging; myocarditis.

Figure 1

Study design. (A) Cardiac function trial: Porcine cardiac myosin was injected subcutaneously (s.c.) in BALB/c mice with CFA (myocarditis group) and without CFA (control group) at day 0 and day 7. Echocardiography to evaluate the left ventricular ejection fraction (EF) was performed on day 0 and day 28. Thereafter, mice were sacrificed for histology. (B) LIBS-MB trial: EAM induction (Myocarditis group) at day 0 and day 7 alongside native echocardiography on day 0 and day 9 with contrast echocardiography using LIBS-MB/control-MB on day 9. (C) Prasugrel therapy trial: EAM was induced at day 0 and day 7 combined with 1 month of oral prasugrel application by daily oral gavage. Native echocardiography was performed on day 0, day 9, and day 28. Contrast echocardiography using LIBS-MB/control-MB was performed on day 9.

Figure 2

LIBS-MB is not suitable for specific detection of early myocarditis by ultrasound in mice. (A) Wash-in of LIBS-MB directly after bolus injection depicted via B-mode on the left and non-linear contrast mode on the right in the parasternal short axis. (B) Residual signal of LIBS-MB after 8 min, when imaging was resumed right before bursting depicted via B-mode on the left and non-linear contrast mode on the right in the parasternal short axis. The left ventricular myocardium is shown as ROI within the green frame and shows no macroscopically visible residual signal. (C) The differential Targeted Enhancement of both LIBS-MB (n = 9) and control-MB (n = 9) in the myocardium of myocarditis mice on day 9 obtained by contrast echocardiography shows no selective binding of either contrast agent (Wilcoxon test, two-tailed, paired). (D) Fluorescence microscopy in 40× magnification, showing bound microbubbles in the myocardium of myocarditis mice on day 9, as exemplarily shown by the white arrow. (E) Counting of bound microbubbles in the myocardium of myocarditis mice on day 9 supports the ultrasound findings: No significant difference between binding of LIBS-MB (n = 6) and control-MB (n = 4) could be observed (Mann–Whitney test, two-tailed).

Figure 3

Platelets occur before inflammatory infiltrates and heart failure; P₂Y12 receptor inhibition with prasugrel limits heart failure and platelet aggregation in the myocardium in murine myocarditis. (A) Comparison of the ejection fractions of mice with myocarditis (n = 11), mice without myocarditis (n = 6), and prasugrel therapy mice (n = 6). On day 28, a significantly improved ejection fraction of the prasugrel therapy group was seen (ordinary one-way ANOVA). (B) Paired observation of the ejection fractions of myocarditis mice with simultaneous oral prasugrel therapy (n = 6) shows a mean ejection fraction on day 28 similar to the baseline level (Wilcoxon test, two-tailed, paired, n.s. (not significant)). At the same time, a widespread distribution is evident with some mice preserving their ejection fraction and some mice deteriorating to the level of myocarditis mice without therapy. (C) Inflammatory infiltrates in HE staining tend to be reduced in prasugrel therapy mice (n = 6) compared to the myocarditis mice on day 28 (n = 11), though the difference is not significant. Myocarditis mice on day 28 exhibit more inflammatory infiltrates than myocarditis mice on day 9 (n = 11) and control mice on day 28 (n = 6, Kruskal-Wallis test, Dunn’s multiple comparison test). (D–F) HE staining in 40× magnification showing a representative image of myocardial tissue from control animals, myocarditis mice, and prasugrel therapy mice on day 28 by light microscopy. (G) Platelet infiltrates in the myocardium of prasugrel therapy mice on day 28 (n = 6) are significantly reduced compared to myocarditis mice both on day 9 (n = 11) and day 28 (n = 11). The counted amounts of platelets are in the range of control mice (n = 6, Ordinary one-way ANOVA, Tukey’s multiple comparisons test). (H) CD41 immunohistochemistry in 40× magnification showing few to no CD41-positive aggregates in healthy myocardial tissue from control mice on day 28 by light microscopy. (I) CD41 immunohistochemistry in 40× magnification showing a high number of CD41-positive aggregates in myocardium from myocarditis mice on day 9 by light microscopy. (J) CD41 immunohistochemistry in 40× magnification showing few to no CD41-positive aggregates in myocardial tissue from prasugrel therapy mice on day 28 by light microscopy.