Ex Vivo and In Vivo Administration of Fluorescent CNA35-Protein Specifically Marks Cardiac Fibrosis

Background Cardiac fibrosis has severe adverse effects on systolic and diastolic function and increases the risk of arrhythmias. To detect cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. In this study, the potential of labeled CNA35, a protein that specifically binds to vascular collagen, is investigated as a specific marker of cardiac fibrosis. Picrosirius red (PSR) staining served as reference for cardiac fibrosis detection. Methods Fluorescently labeled CNA35 (2.5 µM) was applied to frozen tissue sections of dog myocardium (n = 36). After CNA35 quantification, sections were histologically examined with PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 (2 × 38 µM) was administered intravenously in mice (n = 8). Hearts were isolated, and CNA35 labeling was examined in frozen tissue sections (n = 34). Serial sections were examined with PSR. Results CNA35 application on frozen canine myocardium shows specific CNA35 binding to collagen. CNA35 labeling highly correlates with PSR (r = 0.98, P <.001). After in vivo administration in mice, myocardial CNA35 labeling is clearly observed, suggesting that labeled CNA35 is able to penetrate the cardiac endothelium. Strong correlation is observed between CNA35 and PSR (r = 0.91, P <.001; Figure). Conclusions CNA35 specifically binds to cardiac collagen when applied to frozen sections and after in vivo application of CNA35. Strong correlation has been observed between CNA35 and PSR. These data indicate that CNA35 is useful for quantification and localization of myocardial fibrosis and warrants further research into its use for monitoring cardiac fibrosis in vivo. Cardiac fibrosis has severe adverse effects on systolic and diastolic function and increases the risk of arrhythmias. To detect cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. In this study, the potential of labeled CNA35, a protein that specifically binds to vascular collagen, is investigated as a specific marker of cardiac fibrosis. Picrosirius red (PSR) staining served as reference for cardiac fibrosis detection. Fluorescently labeled CNA35 (2.5 µM) was applied to frozen tissue sections of dog myocardium (n = 36). After CNA35 quantification, sections were histologically examined with PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 (2 × 38 µM) was administered intravenously in mice (n = 8). Hearts were isolated, and CNA35 labeling was examined in frozen tissue sections (n = 34). Serial sections were examined with PSR. CNA35 application on frozen canine myocardium shows specific CNA35 binding to collagen. CNA35 labeling highly correlates with PSR (r = 0.98, P <.001). After in vivo administration in mice, myocardial CNA35 labeling is clearly observed, suggesting that labeled CNA35 is able to penetrate the cardiac endothelium. Strong correlation is observed between CNA35 and PSR (r = 0.91, P <.001; Figure). CNA35 specifically binds to cardiac collagen when applied to frozen sections and after in vivo application of CNA35. Strong correlation has been observed between CNA35 and PSR. These data indicate that CNA35 is useful for quantification and localization of myocardial fibrosis and warrants further research into its use for monitoring cardiac fibrosis in vivo.