The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis

Abstract

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Extracellular Cyclophilin A is associated with myocardial fibrosis.

A, Representative stainings of heart sections from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Sections were stained with Masson’s Trichrome, anti-CyPA, and IgG-control as indicated. Marked area is magnified in the middle panel. B, Representative image of healthy control mice stained with anti-CyPA or IgG control. C, Myocardial stainings from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Myocardial sections were stained with anti-CyPA (green), rhodamin phalloidin (red) for actin cytoskeleton, and ToPro-3 for nuclei (blue), as described in materials and methods. Arrows indicate localization of CyPA in the extracellular space.

Fig 2. Extracellular Cyclophilin A—inhibition by MM284 attenuates migration of human monocytes in vitro.

A, Effects of MM284 on Cyclophilin A-induced chemotaxis were studied using a modified Boyden chamber. Migration of human monocytes was assessed using media, SDF-1α (50ng/ml) as positive control, CyPA (200nM), or CyPA (200nM) + MM284 (200; 500; and 800nM). Cells were incubated for 4h at 37°C. Migrated cells were counted and a chemotactic index was calculated (n = 5). B, Adhesion of monocytes under flow conditions to activated human umbilical vein endothelial cells. Data are shown as mean ± SEM. C, Cell membrane permeability of MM284 was assessed using a competition assay. MM284 or NIM811 (a cell permeable CsA derivate) was used to displace Fluo-mCsA (a fluorescently labeled (green), cell permeable CsA derivate) from the cytoplasm of THP1 cells. The presence of Fluo-mCsA in the cytoplasm after treatment was assessed using confocal laser scanning microscopy. * indicates p < 0.05 compared to CyPA 200nM.

Fig 3. MM284 reduces cardiac inflammation and fibrosis in autoimmune myocarditis in mice.

Troponin I-induced autoimmune myocarditis was studied in A/J mice. A, Hematoxylin and Eosin staining (H&E) was used to determine the area of cardiac damage 28 days after induction of myocarditis. B, Masson’s Trichrome staining was used to determine the extent of fibrotic remodeling in the myocardium 28 days after induction of experimental autoimmune myocarditis. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05. C, 28 days after induction of autoimmune myocarditis, small animal echocardiography was used to evaluate left ventricular function (n = 7). Left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular mass (LV Mass), left ventricular end-systolic volume (LV ESV), left ventricular end-diastolic volume (LV EDV), left ventricular posterior wall in diastole (LVPWd), heart rate, and body weight are shown. n.s. indicates not significant.

Fig 4. MM284 reduces infiltration of T-cells and macrophages in autoimmune myocarditis in mice.

A, B, Infiltration of T-cells and macrophages was assessed using anti-CD3 and anti-Mac-3 staining. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05.

Fig 5. Influence of MM284 on myocardial TNFα, IL-6 and MMP-9 expression.

Realtime PCR assay was performed to evaluate expression of TNFα (A), IL-6 (B) and MMP-9 (C) in the myocardium. Data are shown as mean of relative expression ± SD, n ≥ 8. * indicates p < 0.05, n.s. indicates not significant.