Targeted cardiac inactivation of Guanylate binding protein 5 (GBP5) protects from cardiotoxicity after radiation

Background Thoracic radiotherapy is still the treatment of choice for many oncological diseases. Despite state-of-the-art radiation techniques, patient-centred and personalized radiation of the tumours, patients who undergo thoracic radiotherapy have a significantly increased risk of cardiac events and cardiac fibrosis compared to the non-irradiated comparison cohort. While the damaging effect of photon irradiation is well documented, the specific molecular mechanisms leading to cardiac fibrosis is still unknown. In our previous work, we identified GBP5 (Guanylate-binding protein 5) as a radiation-specific gene, upregulated in vitro and in vivo after radiation. GBP5 plays a crucial role in the assembly of the NLRP3 inflammasome, a complex responsible for triggering the release of pro-inflammatory cytokines, including IL-1β and IL-18. However, its precise role in radiation-related cardiac inflammation which finally leads to cardiac fibrosis remains poorly understood. Methods We generated an inducible, conditional, cardiomyocyte-specific knock-in mouse, expressing a dysfunctional GBP5 molecule (GBP5-cKI), lacking the c-terminus which is necessary for NLRP3 assembly. To explore its role in cardiac fibrosis after photon irradiation, mice were irradiated with 16 Gray photon and sacrificed after 2 weeks and 5 months to analyze the acute and chronic cardiac inflammation after radiation. Results Mice carrying the dysfunctional GBP5 knock-in are significantly protected against radiation-induced mortality. GBP5 knock-in mice show a markedly prolonged survival compared to wild-type controls (p=0,0276). Cardiac function was largely preserved after 5 months in treated knock-in animals (FS 26,92 %± 1,111 vs. 23,01 %±1,550 GBP5-cKI vs. WT controls; p= 0,0001; EF 53,14 %±1,691 vs. 46,86 ± 2,614 GBP5-cKI vs. WT controls; p <0.0001; GLS -17,75%±0,4338 vs. -12,60±0,7667 GBP5-cKI vs. WT controls; p <0.0001; measured echocardiographically in anaesthetic animals). Knock-in mice showed 5 months post radiation lower levels of Caspase-1 and IL1b activation (1,889 ± 0,2805 vs. 2,448 ± 0,08422 GBP5-cKI vs. WT controls; p <0.0001; 2,105 ±0,3705 GBP5-cKI vs. WT controls; p= 0,0022; qPCR results were normalized to 18S expression, results symbolize fold change) and significantly reduced production of fibrose related genes (CTGF 1,057±0,1785 vs. 1,622 ± 0,5396 GBP5-cKI vs. WT controls; p= 0,1194; CCL2 1,417± 0,2592 vs. 1,896 ± 0,2586; GBP5-cKI vs. WT controls; p= 0,0165; qPCR results were normalized to 18S expression, results symbolize fold change), indicating disruption of the NLRP3 activity leads to reduced cardiac fibrosis. Similar findings were observed on protein levels. Implications These results suggest that the C-terminal GBP5 modulates the inflammasome pathway and is for myocardial inflammation after radiation essential. GBP5 is a potential therapeutic target to reduce cardiac damage after radiation.