Cardiomyocyte-derived BDNF restricts cardiac fibrosis by decreasing the activity of the TGF-β/Smad2/3 pathway and increasing Smad7 expression

Background and objective To investigate the role of cardiomyocyte-derived BDNF as an endogenous regulator to decrease cardiac fibrosis, its underlying mechanism and therapeutic potential. Methods Single-nuclei RNA sequencing (snRNA-seq), KEGG, Gene Ontology and cell‒cell interaction analyses were performed to identify changes in cardiac cells, cardiac functions and pathways due to the conditional knockout of cardiomyocyte-derived BDNF (cardiomyocyte-BDNF-KO). Protein C-terminal sequencing, qPCR, WB, CCK8 assays, flow cytometry, BDNF-AAV9 treatment and histological staining were performed to investigate the roles of BDNF and the BDNF mimic 7,8-DHF (7,8-DHF) in cardiac fibroblasts cardiac myofibroblasts and cardiac fibrosis, and the cross-inhibition of the TGF-β and BDNF–TrkB-FL pathway. Results snRNA-seq and bioinformatics analysis revealed that cardiomyocyte-BDNF-KO significantly increased the percentage of CFs, decreased the number of cardiomyocytes, and increased the activity of the TGF-β pathway in CFs. Functional studies confirmed that compared with those in wild-type hearts, the expression levels of key signaling molecules in the TGF-β pathway in cardiomyocyte-BDNF-KO CFs in the mouse heart were significantly higher. CFs and CMFs expressed the BDNF receptor TrkB-FL but not BDNF, and treatment with BDNF and 7,8-DHF decreased the expression of key signaling molecules in the TGF-β pathway in CFs and CMFs. BDNF inhibited CF and CMF proliferation, inhibited CF activation and transformation into CMFs, promoted CMF apoptosis, the accumulation of cells in S phase of the cell cycle and TrkB-FL phosphorylation in CFs and CMFs, increased Smad7 expression in CMFs, and inhibited the activity of the TGF-β/Smad2/3/α-SMA pathway. 7,8-DHF had the same effects as BDNF, as documented above. Furthermore, BDNF-AAV9 therapy for cardiomyocyte-BDNF-KO hearts increased Smad7 expression and decreased the activity of the TGF-β/Smad2/3 pathway and the expression of fibrotic effectors, which ameliorated cardiac fibrosis. Conclusion Cardiomyocyte-derived BDNF acts as an endogenous mediator to restrict cardiac fibrosis by inhibiting CF and CMF proliferation, CF activation and transformation into CMFs, and increasing arrest in S phase of the cell cycle in CFs and CMFs and the apoptosis of CMFs. The BDNF–TrkB-FL pathway cross-inhibits the activity of the TGF-β/Smad2/3/α-SMA pathway and increases the expression of Smad7. BDNF and 7,8-DHF have therapeutic potential for treating cardiac fibrosis.