Background: Although myocarditis is a rare complication of rheumatic diseases such as lupus and rheumatoid arthritis, mortality resulting from it may be as high as 20%. We hypothesized that the clinical signs of sudden onset tachycardia and/or heart failure, are preceded by dysregulation at tissue level, where persistent, widespread inflammation contributes to continued tissue damage. More specifically, fibrosis - which forms part of the tissue repair process – is excessive in rheumatic disease. This process, which is promoted by transforming growth factor (TGF-β), leads to progressive architectural changes and dysfunction in organs, to the point of organ failure. However, bone morphogenetic protein-7 (BMP-7) has been shown to counter the effects of dysregulated TGF-β signalling in the context of rheumatoid cachexia as well as non-rheumatic disease models. This poses potential for preventative intervention in the context of rheumatic cardiac disease. Based on the ability of BMP-7 to regulate the processes of inflammation and fibrosis, therapeutic modulation of the TGF-β:BMP-7 ratio may benefit rheumatic disease management.

Objectives: Using a preclinical, in vivo (zebrafish) model of myocarditis, the current study aimed to determine the extent to which different (traditional and experimental) treatments may alter the TGF-β:BMP-7 ratio and fibrosis outcome.

Methods: Following ethical approval of protocols, a sclerosing agent was micro-injected into the pericardial space of larval zebrafish, at 2 days post fertilization (dpf). Sham-injected larvae were injected with phosphate buffered saline (PBS) only. Larvae were subsequently maintained in media containing either a placebo, or one of 25 µg/ml prednisolone, 28.57 µg/ml hydro chloroquine (HCQ) – both human equivalent doses - or 750 ng/ml BMP-7 (literature-based effective experimental dose), for a period of 60 hours. At experimental endpoint at 4.5 dpf, larvae were euthanised, fixed and fluorescently labelled for TGF-β, BMP-7 and the fibroblast marker vimentin.

Results: Following injection with a sclerosing agent, larval hearts exhibited increased TGF-β expression (p<0.01 vs control), which was partially rescued by all treatments. Furthermore, HCQ treatment was associated with a significant increase in BMP-7 expression in the cardiac region (vs control and prednisolone, both p<0.05). A similar outcome was observed after BMP-7 treatment (vs control, p=0.05), while prednisolone did not affect BMP-7 expression. In terms of the TGF-β:BMP-7 ratio, injection alone and prednisolone treatment elevated the ratio, whereas treatment with both HCQ and BMP-7 normalised the ratio.

Conclusion: Current data validates our approach of simulating myocarditis via injection of a sclerosing agent into the pericardial space of zebrafish larvae. Furthermore, data suggest that treatment with both HCQ and BMP-7 effectively increase BMP-7 to normalise the TGF-β/BMP-7 ratio, demonstrating potential in the treatment of myocarditis. Given the known long-term side-effects of HCQ, BMP-7 should be evaluated as alternative anti-fibrotic treatment in rheumatology patients.

REFERENCES: [1] Du Toit R, Karamchand S, Doubell AF, Reuter H, Herbst PG. Lupus myocarditis: review of current diagnostic modalities and their application in clinical practice. Rheumatology (Oxford). 2023. doi: 10.1093/rheumatology/keac409.

[2] Smith C, du Toit R, Ollewagen T. Potential of bone morphogenetic protein-7 in treatment of lupus nephritis: addressing the hurdles to implementation. Inflammopharmacology. 2023 doi: 10.1007/s10787-023-01321-x.

[3] Ollewagen T, Tarr GS, Myburgh KH, Reuter H, Smith C. Therapeutic Benefit in Rheumatoid Cachexia Illustrated Using a Novel Primary Human Triple Cell Coculture Model..Int J Inflam. 2022. doi: 10.1155/2022/1524913.

Acknowledgements: NIL.

Disclosure of Interests: None declared.