TRPV4-associated proinflammatory macrophage activation and cardiomyocyte ferroptosis-related injury in myocardial ischemia/reperfusion: involvement of USP47/Wnt/β-catenin signaling

Background Mixing oxidative damage, sterile immunological responses, and controlled cell-death pathways causes myocardial ischemia-reperfusion injury (MIRI). Although MIRI is related with macrophage diversity and ferroptotic damage in cardiomyocytes, it is unknown how inflammatory macrophage states cause ferroptosis-associated cardiac harm. Our study investigated the relationship between TRPV4-induced macrophage activation, USP47/Wnt/β-catenin activity, and cardiomyocyte damage caused by macrophages. Methods A public single-cell RNA-sequencing dataset was reanalysed to characterise MIRI macrophages and cardiomyocytes. Experimental clinical samples and RAW264.7 macrophage and HL-1 cardiomyocyte tests supported these results. TRPV4 was repressed with shRNA and Wnt/β-catenin and JAK2/STAT3 signalling were pharmacologically inhibited. Flow cytometry, cytokine tests, and protein markers assessed macrophage polarisation. LPS/IFN-γ-polarized macrophages’ conditioned media was given to HL-1 cells, with or without N-acetylcysteine or ferrostatin-1. Both biochemical and protein indicators were used to evaluate cardiomyocyte damage and ferroptosis. Results In MIRI, single-cell data indicated substantial macrophage rearrangement, including growth of a Trpv4-enriched subgroup with proinflammatory transcriptional state, whereas multiple cardiomyocyte subsets had greater ferroptosis-related signals. In RAW264.7 cells, TRPV4 knockdown reduced F4/80 + CD86 + fraction, iNOS, and proinflammatory cytokines, while increasing IL-10 and TGF-β. It also restored USP47 and phospho-β-catenin. Wnt/β-catenin signalling inhibition partially reduced the anti-inflammatory effects of TRPV4 silencing, suggesting its functional involvement. NAC and Fer-1 partly corrected cardiomyocyte damage, lipid-peroxidation- and iron-associated indices, GPX4 and SLC7A11 changes, and ACSL4 elevation in M1-like macrophage-conditioned media. AG490 decreased LPS-induced inflammatory polarisation and JAK2/STAT3 activation. Conclusions TRPV4-linked inflammatory activation of macrophages, along with reduced USP47/Wnt/β-catenin signalling, promotes paracrine, ferroptosis-associated cardiomyocyte injury during MIRI. JAK2/STAT3 is better viewed as a parallel inflammatory amplifier rather than a downstream element of the TRPV4 pathway. This multicellular model needs mechanistic and in vivo testing before immunomodulatory and anti-ferroptotic treatments may be developed.