Exosomal miR-124 from M2 macrophages protects against hypertensive cardiac remodeling through suppression of Calpain-1

BACKGROUND: While M2 macrophages are known to facilitate tissue repair, the specific paracrine mechanisms by which they modulate cardiomyocyte responses during hypertensive cardiac remodeling remain largely undefined. This study aimed to elucidate the role of M2 macrophage-derived exosomes (M2-exo) in regulating cardiac homeostasis under hypertensive stress and to identify the specific molecular cargo responsible for these effects. METHODS: Exosomes were isolated from IL-4/IL-13-stimulated mouse peritoneal macrophages (M2-exo) and characterized via transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. The therapeutic potential of M2-exo was evaluated in Angiotensin II (Ang II)-infused hypertensive mice and in primary neonatal rat cardiomyocytes and fibroblasts in vitro. The underlying molecular mechanisms were dissected using miRNA sequencing, luciferase reporter assays, and AAV9-mediated gene transfer. RESULTS: Systemic administration of M2-exo markedly attenuated Ang II-induced cardiac hypertrophy and fibrosis in mice. In vitro, M2-exo suppressed fibrotic gene expression in cardiac fibroblasts and reduced hypertrophic growth in cardiomyocytes. miRNA profiling revealed enrichment of microRNA-124 (miR-124) in M2-exo compared to M0-exo. AAV9-mediated overexpression of miR-124 recapitulated the protective effects of M2-exo in vivo. Capn1, encoding Calpain-1, was identified as a direct target of miR-124. Both pharmacologic inhibition of Calpain-1 (MDL28170) and AAV9-mediated overexpression of calpastatin, an endogenous Calpain inhibitor, improved cardiac remodeling. However, these interventions did not further enhance the effects of M2-exo. CONCLUSION: Our study uncovers a novel immunomodulatory axis wherein M2 macrophages mitigate hypertensive cardiac remodeling via the exosomal transfer of miR-124. By directly targeting Capn1 to suppress deleterious Calpain-1 activation, the M2-exo/miR-124/Calpain-1 axis preserves myocardial structural integrity, presenting a precise therapeutic target for hypertensive heart disease.