Age-associated elevated inflammation and immune pathways in the mice heart are linked to Adiponectin-Adipor1-RelA signaling

Inflammatory gene profiles using RNA seq analysis were studied by measuring pro-inflammatory cytokines and chemokines levels. qRT-PCR and Western blot analysis were used to validate the expression profile of these inflammatory mediators. Using flow cytometry analysis, CD11b+ monocytes and CD64+ Ly6C were quantified in the young and old hearts. The inflammatory response, Adipor1 and Adipor2 gene expression, RelA nuclear translocation and the effects of adiponectin in LPS-stimulated or Adipor1 silenced H9C2 cells were studied. Gene ontology analysis using differentially expressed genes revealed an enrichment of immune response pathways in the old mice hearts when compared to young mice hearts. Western blot analysis confirmed the down regulation of several anti-inflammatory proteins and the upregulation of pro-inflammatory proteins including CD68, NF-kB1 and Rel-A, in the old mice hearts. Flow cytometry suggested an infiltration of CD11b+monocytes and CD64+ Ly6C-high macrophages in the old mice hearts compared to younger hearts confirming an increased inflammation in the older hearts. Mechanistically, to understand if the Adiponectin-Adipor1-NFkB axis regulates inflammation in the aging heart, Adipor1 and Adipor2 genes were silenced in H9c2 cardiomyocytes. Immune response genes were elevated in the Adipor1 silenced H9c2 cells but not in Adipor2 silenced cells. Pretreatment with Adiponectin (APN) attenuated the Adipor1 silenced or lipopolysaccharides (LPS)-stimulated expression of inflammatory genes in H9c2 cardiomyocytes. APN also attenuated the nuclear translocation of RelA and induction of immune response genes in Adipor1 silenced or LPS-challenged H9c2 cardiomyocytes. APN-AdipoR1-RelA signaling might be a novel therapeutic target for the treatment of inflamed elderly hearts.