MLKL–Mediated Necroptosis Predominantly Contributes to Immune-Associated Myocardial Damage

Activated T cells and macrophages play a critical role in immune-associated myocarditis. However, the molecular and cellular mechanisms driving cardiomyocyte damage by immune cells remain poorly understood. In this study, we co-cultured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with activated human peripheral blood mononuclear cells (aPBMCs) to recapitulate myocardial infiltration of immune cells. Our results demonstrated that aPBMCs induced hiPSC-CMs death in a dose- and time-dependent manner. Transcriptome analysis revealed the activation of several death pathways, including pyroptosis, apoptosis and necroptosis. The time course of immunofluorescence staining of key proteins related to different death pathways demonstrated that necroptosis was the earliest activated pathway. Pharmacological blockade of necroptosis by targeting mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein RIPK1 kinase 3 (RIPK3) protected hiPSC-CMs against injury induced by aPBMCs, while inhibitors of pyroptosis and apoptosis showed no protective effect. Moreover, MLKL knockdown in hiPSC-CMs prevented cell death due to aPBMCs challenge. Additionally, we validated the cardioprotective effects of blocking necroptosis in a mouse model of immune checkpoint inhibitors (ICIs)-related myocarditis using a combination of long-term anti-programmed cell death 1 (PD- 1) and anti-cytotoxic T-lymphocyte antigen- 4 (CTLA- 4) antibodies. ICIs led to elevation of myocardial injury markers in serum and activated immune cells infiltration. Furthermore, in vivo administration of a MLKL inhibitor prevented ICIs-induced myocardial injury. In conclusion, our findings suggested that MLKL-mediated necroptosis predominantly contributed to cardiomyocyte death resulting from activated immune cells. Suppressing necroptosis may be an effective therapeutic approach against myocardial damage in myocarditis.