Clin Res Cardiol (2021) DOI DOI https://doi.org/10.1007/s00392-021-01843-w

Cardiac plakophilin-2 knockout mice develop progressive biventricular cardiomyopathy with inflammatory response

T. Williams1, A. Kilinc1, R. Groß1, S. Schraut1, M. Delgobo1, G. Ramos1, A. P. Arias Loza1, B. Gerull1

1Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg;

Background: Mutations in the desmosomal gene plakophilin 2 (PKP2) gene have been identified in arrhythmogenic cardiomyopathy (ACM) patients. In cardiac muscle PKP2 is found in desmosomal structures located within intercalated discs, where it functions to link cadherins to intermediate filaments in the cytoskeleton, thereby providing functional and structural integrity. However, the molecular and cellular events underlying PKP2 dysfunction involved in the pathogenesis of the disease remain elusive. Methods and Results:  We are using cardiac restricted Pkp2 knockout mice to investigate the role of Pkp2 in ACM. qRT-PCR and Western blot analysis demonstrated a significant (-90%) decrease in cardiac Pkp2 mRNA and protein levels. Morphological and histological analyses of animals using Hematoxylin/Eosin and Picrosirius Red staining indicated that at 8 weeks of age Pkp2 knockout animals exhibit progressive loss of cardiac myocytes, extensive inflammatory infiltration and fibrous tissue replacement in both ventricles, with an early hit on the RV as seen in ACM patients. Cardiomyocytes of Pkp2-/- mice were hypertrophic as evidenced by an enlarged cross-sectional area (+12%, p<0.001). Echocardiography showed onset of mild LV dilation with LV systolic and RV dysfunction in these animals at 4 weeks of age, which was more pronounced at 8 weeks as seen by an increase in the LV enddiastolic diameter (+13%, p<0.05) paralleled by significantly decreased fractional shortening (-42%, p<0.01), ejection fraction (-37%p<0.0001), fractional area change (-34%, p<0.01), and significantly reduced tricuspid annular plane systolic excursion (TAPSE, -43%, p<0.01). It has been proposed that the presence of inflammatory infiltrates is a major component of human ACM disease initiation and progression. We therefore performed flow cytometry analysis to determine and define cardiac immune cells and their subpopulations in our animal model. At 8 weeks there was a significant difference in CD45+ cells (2.5-fold, p<0.01) in the Pkp2-/- heart compared to age matched controls, especially regarding the CD11b+ population (2.5-fold increase, p<0.01). Cardiac B- and T-Cells were also altered as seen in an increase in CD19+ (2.3-fold, p<0.05) and total CD3+ cell counts (3-fold, p<0.05), respectively. Immunohistochemistry to capture the specific cell types confirmed that a multitude of infiltrating cells in the Pkp2-/- hearts consisted of macrophages. Finally, at the age of 8-12 weeks, we detected cardiac specific auto-antibodies by immunofluorescence in the sera of our Pkp2-/- mice as seen in the human disease phenotype of myocarditis/DCM, which were absent in control littermates. Outlook: Our data suggest that mice with a cardiac tissue-restricted loss of Pkp2 develop progressive biventricular cardiomyopathy with focal areas of cardiac fibrosis, inflammatory infiltration and cardiac dysfunction as seen in human ACM patients. In addition, we detect cardiac-specific autoantibodies in our mouse model, a shared feature of myocarditis. All these observations provide strong evidence that inflammation is a major “driver” of disease progression in our animal model. Nevertheless, a more detailed analysis of these first findings remain to be the subject of further studies, especially with regards to underlying molecular mechanisms.

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