Objective

2

To investigate the effects of programmed cell death protein⁃1 (PD⁃1) monoclonal antibody on left ventricular myocardial inflammation

myocardial fibrosis

and cardiac function in myosin⁃induced autoimmune myocarditis mice

and to establish a murine model of immune checkpoint inhibitor (ICI)⁃associated myocarditis.

Methods

2

Eighteen male BALB/c mice were randomly divided into three groups: normal control group

experimental autoimmune myocarditis (EAM) group

and EAM+PD⁃1 inhibitor group. Mice in the EAM and EAM+PD⁃1 inhibitor groups received subcutaneous immunization with 100 μg mouse heavy chain myosin (Myhc⁃α) emulsified with complete Freund's adjuvant at a 1∶1 ratio on days 0 and 7. From day 8

the EAM group was administered isotype IgG intraperitoneally at 5 mg·kg

-1

per dose

and the EAM+PD⁃1 inhibitor group received PD⁃1 inhibitor at 5 mg·kg

-1

per dose intraperitoneally every three days

for a total of five doses. After 21 days of treatment

echocardiography was performed

and ELISA was used to measure circulating cardiac injury markers (cTnI) and inflammatory cytokines (IFN⁃γ and TNF⁃α) levels. Left ventricular myocardium was evaluated for pathological changes and inflammatory cell infiltration using HE staining and immunohistochemistry. Myocardial fibrosis was assessed via Masson staining.

Results

2

Compared to the control group

EAM mice showed significantly reduced LVEF and increased LVESD. The reduction in LVEF was even more pronounced in the EAM+PD⁃1 inhibitor group than in the EAM group

with a more sign

ificant increase in LVESD. HE staining and immunohistochemistry staining revealed inflammatory cell infiltration in the EAM group

which was more severe in the EAM+PD⁃1 inhibitor group. Masson staining demonstrated a substantial increase in myocardial collagen fibers in the EAM group

with elevated collagen volume fraction

which was further exacerbated in the EAM+PD⁃1 inhibitor group than in the EAM group. ELISA results indicated elevated peripheral blood levels of cTnI

TNF⁃α

and IFN⁃γ in EAM mice compared to controls

with the highest levels observed in the EAM+PD⁃1 inhibitor group (

P

<

0.05).

Conclusion

2

In the mice with Myhc⁃α⁃induced myocarditis

combined with multiple intraperitoneal injections of PD⁃1 monoclonal antibody

the myocardial inflammatory cell infiltration

myocardial injury

myocardial fibrosis

and decline in cardiac function were exacerbated. This experiment successfully established a mouse model of ICI⁃associated myocarditis.