Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Cardiac Troponin I antibodies mediate cardiomyocyte damage and alter morphology

J. Furkel1, V. Zirkenbach1, M. Knoll2, N. Frey1, M. Konstandin1, Z. Kaya1

1Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg; 2Abteilung für Radioonkologie und Strahlentherapie, Universitätsklinikum Heidelberg, Heidelberg;

Background Development of myocarditis has multiple etiologies and represents an important cause of heart failure in young adults. It is defined as an inflammatory disease of the heart muscle and identified by histological, immunological and immunohistochemical criteria. Recently, it has been shown that immunization with cardiac troponin I induces severe myocardial inflammation, fibrosis and heart failure in mice. Furthermore, cardiac troponins entering the circulation after cardiac damage lead to the production of autoantibodies. However, the role of autoantibodies and the molecular mechanisms remain largely unknown and clinical risk stratification is challenging. Therefore, the aim of this study was to functionally evaluate the direct effects of cardiac troponin I autoantibodies to the cardiomyocyte. Methods 5-week-old female A/J wild-type mice were immunized twice with 150µg murine cardiac Troponin I (mcTnI). One group of control mice were immunized only with buffer solution the second control group were untreated mice. Serum was collected from all mice after 21 days. To detect myocardial damage, we measured hsTnT levels and antibody titer against mcTnI in mouse sera. Cardiac heart function was controlled by echocardiography and inflammation checked by histopathological staining of heart sections.  To study the direct effect of autoantibodies on cardiomyoctes neonatal rat cardiomyocytes were incubated for 48h with collected sera (5%, 20%) and morphological changes were detected using the recently published C-MORE assay. Dimension reduction of single cell data was achieved using the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) method. Morphological findings (cell death, cell area) were validated in heart sections via manual cell size assessment and TUNEL staining. Results Immunization with mcTnI leads to elevated hsTnT levels (mean=1504±670.3,n=5;p=0.0882) compared to buffer immunized or untreated mice (mean=0±0, n=5). Cardiac function showed reduced ejection fraction after 21 days (mean=68.9;p<0,0001) compared to day 0 (mean=82.21). Evaluation of inflammation score showed inflammatory areas in TnI immunized mice (mean=45±11.73,n=5;p=0.0185) but no inflammation in control groups. Incubation of cardiomyocytes with mouse serum showed a concentration dependent morphological change in mcTnI mice. Cell cycle analysis revealed an increased portion of apoptotic cardiomyocytes for 20% sera (lin. model p<0.001), but not for 5%. This was validated in heart tissue sections (TUNEL, median=13,91, n=6;p=0.0022). Additionally, in mcTnI groups we found an increase in cell size in vitro for 5% serum (lin. model p<0.05), but showed a decrease for 20% serum (lin. model,p <0.001). In vivo we observed an increase cell size (øTnI=73,14±1,04,n=61 vs. øCFA=67.63±0.7231.n=33;p=0.0004). Single cell analysis of the vital cardiomyocyte fraction shows a distinct morphological population for mcTnI containing three main subpopulations. Conclusion: In this study we demonstrated that mouse serum containing autoantibodies against mcTnI can alter cell morphology of cardiomyocyte. Our results hint towards a direct causal effect of the circulating autoantibodies to cardiomyocytes with a concentration dependent damage of cardiomyocytes. Therefore therapeutic agents targeting antibodies against mcTnI or their interaction may be attractive options.

https://dgk.org/kongress_programme/jt2022/aP1887.html