Fig 2: Knockdown of Bhlhe40 by rAAV9-shBhlhe40 injection inhibits Ang II-induced atrial fibrosis. (A) Representative Masson staining for fibrosis that stained in blue (left) and quantification of the fibrotic area of the Masson-stained sections from the rAAV9-shCON-treated/rAAV9-shBhlhe40- treated and Ang II-infused/saline-infused hearts (right; n = 6 mice per group). (B) Representative IHC and quantification of a-SMA-positive cells (right; n = 6 mice per group). (C,D) qPCR analyses of collagen I and collagen III mRNA levels in Ang II-induced atrial tissues. (E) Immunoblotting analyses of TGF-ß1, p-Smad2, and Smad2 protein levels in atrial tissues (left), and the quantification of each protein (right, n = 4). n indicates the number of animals used in each group. *P < 0.05, **P < 0.01.

Fig 3: Working model of the effect of knockdown of Bhlhe40 on Ang II-induced AF. Cardiac-specific knockdown of Bhlhe40 suppresses Ang II-induced atrial inflammation, atrial fibrosis, electrical remodeling, and signaling pathways (NF-?B, NLRP3/IL-1ß, TGF-ß/Smad2, Kv4.3, and CX43) in mice.

Fig 4: Knockdown of Bhlhe40 by rAAV9-shBhlhe40 injection reduces Ang II-induced atrial electrical remodeling. (A) Representative immunofluorescence for Cx43 (red) in atrial tissues from Ang II-infused or saline-infused mice. (B) Quantification of the intensity of CX43 expression in atrial tissues. (C) Immunoblotting analyses (left) and quantification of ion channel subunits and connexin (Kv4.3and CX43) in atrial tissues (n = 4). Tubulin was used as an internal control. n represents the number of animals (n = 4). *P < 0.05, **P < 0.01.

Fig 5: Expression of Bhlhe40 was significantly upregulated in Ang II-induced atrial tissues and atrial myocytes. (A) qPCR analysis of the mRNA level of Bhlhe40 in Ang II-infused or saline-infused atrial tissues (n = 6). (B) Immunoblotting analysis of Bhlhe40 expression in atrial tissues from each group (upper), and quantification of the Bhlhe40 level (lower, n = 4). (C) IHC staining of Bhlhe40 (left) and the quantification of the Bhlhe40-positive area (right) in atrial tissues from Ang II-infused or saline-infused mice (n = 6). Scale bar = 50 µm. (D) primary atrial cardiomyocytes (ACMs) were treated with Ang II (100 nM) or Saline for 24 h. Bhlhe40 protein levels in ACMs were detected by immunoblotting analysis (left) and quantified (right) (n = 4). **P < 0.01 vs saline-treated WT mice.