1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland
Background: Myocarditis is an inflammatory disease of the heart and a common cause of sudden cardiac death in young adults. Therapeutic approaches are still limited and based on conservative therapy. Therefore, it is necessary to further understand the underlying pathomechanism. Our preliminary work indicates a potential for PNUTS (phosphatase nuclear targeting subunit) in inflammatory cardiomyopathy. PNUTS is part of the protein phosphatase 1 (PP1) that shows an inhibitory effect and directs PP1 into the nucleus. As part of the DNA damage response, PNUTS is targeted to damage sites during G2 checkpoint arrest. It was reported, that a PNUTS overexpression leads to an increased hypoxia-induced cell death with increased levels of Bax. The aim of this study was to further elucidate the role of PNUTS in myocarditis.
Methods: 6-weeks-old AJ mice were transfected with an AAV9-virus construct for overexpression of PNUTS or as a control eGFP in the heart. To study the effect of PNUTS overexpression in our established model for experimental autoimmune myocarditis (EAM), we induced an EAM by weekly immunization with TnI for three times and AAV-transfection 14 day prior. Heart function and inflammation score was investigated by measuring ejection fraction via echocardiography and inflammation score was quantified by histopathological staining. In Addition, hsTnT levels were evaluated to detect myocardial damage and inflammatory markers were investigated on molecular level. Based on the AJ results, 6-weeks-old BL6 mice and BL6 PD-1 ko mice were treated and analyzed the same way as the AJ mice to study the effect of PNUTS overexpression in BL6 mice resistant against an EAM induced by cTnI and in mice used for cardio-oncological studies (BL6 PD-1 ko).
Results: We found an enhanced inflammation in Pnuts overexpressing AJs compared to eGFP controls (43.25±9,846 vs. 20.54±6,247; p=0.053)(Fig. 1A). Interestingly, the sole overexpression of PNUTS in mice treated with control buffer led also to an inflamed heart compared to eGFP control buffer mice (22.75±5.061 vs. 0±0; p<0.001). These findings were accompanied with a reduced cardiac function and elevation of hsTnT levels. The molecular analysis showed elevated inflammatory markers in Pnuts overexpression compared to eGFP. This finding suggests that PNUTS promotes inflammation in the heart. In the next step, we investigated Pnuts overexpression in BL6 PD-1 ko mice and in wildtype BL6 mice. Interestingly, in BL6 PD-1 ko mice Pnuts overexpression led to an increase in inflammation grade (Fig. 1B). A percentual evaluation in these heart sections was not possible. These mice also had elevated hsTnT levels. In contrast, BL6 mice overexpressing Pnuts showed no induction of inflammation in the hearts (Fig. 1C) accompanied by a normal heart function.
Fig.1: Investigation of inflammation after Pnuts overexpressing in (A) AJ, (B) BL6 PD-1 ko and (C) BL6 mice. (A) and (C) were evaluated as percentage, whereas (B) was scored in grades.
Conclusion: This study shows that PNUTS overexpression in the model of EAM enhances inflammation in the heart and induces a pro-inflammatory environment on molecular level. This effect was seen in Pnuts overexpressing AJ mice, but not in resistant BL6 animals. Here, PNUTS overexpression was not sufficient to inudce a myocarditis, while in PD-1 ko BL6 mice overexpressing Pnuts a similar trend could be observed as in AJ mice. The Pnuts effects are therefore strain-dependent.