Abstract Body (Do not enter title and authors here): Introduction: Cardiac fibrosis is a major contributor to adverse cardiac remodeling that underlies heart failure. Previously, we identified the presence of the histamine-3 receptor (H₃R) in the left ventricle (LV), including on fibroblasts, and demonstrated that exogenous H₃R activation with imetit reduced cardiac fibrosis in angiotensin II (Ang II)-infused mice.
Objective: We sought to determine the extent to which activation of H₃R improved diastolic function, and to identify cellular mechanisms by which H₃R opposes a profibrotic cardiac fibroblast phenotype.
Methods: Eight-week-old male C57/B6 mice received saline or Ang II (1500 ng/kg/d) via osmotic pump for 7 days. Separate saline or Ang II mice also received subcutaneous injections of imetit (40 mg/kg/d). LV function was assessed by pressure-volume catheter. TruPath assay was used to identify H₃R-induced G proteins in HEK293T cells. For signaling studies, primary LV fibroblasts were isolated from C57/B6 mice and treated with TGF-β1 (30 ng/mL) as a profibrotic stimulus, together with imetit (300 nM). Collagen release and cell migration were assessed for fibroblast phenotype. The cAMP/PKA/CREB signaling pathway was also assessed.
Results: LV end-diastolic pressure was increased in Ang II mice (p<0.03 vs Saline). This was prevented by imetit (p<0.02 vs Ang II). There was also a trend towards an increase in tau, a measure of LV stiffness, in Ang II mice that was normalized by imetit. Cardiac fibrosis occurred in Ang II mice (p<0.0001 vs Saline), which was normalized by imetit (p<0.0001 vs Ang II). In cardiac fibroblasts, TGF-β1 increased collagen I release, with this being inhibited by imetit (TGF-β1 594.1±59.97 vs TGF-β1+Imetit 274±38.2 ng/mg, p<0.0001). TGF-β1 inhibited fibroblast migration, with imetit restoring a normal migratory response (p<0.003 vs TGF-β1). H₃R activation induced the Gα_i subunit in HEK293T cells, which would suggest inhibition of cAMP. Consistent with this, imetit significantly decreased both pPKA/PKA and pCREB/CREB ratios in cardiac fibroblasts following TGF-β1. The cAMP pathway inhibitor cAMPs-Rp reduced TGF-β1 induced collagen production and restored the cell migratory response lost due to TGF-β1.
Conclusions: Activation of H₃R improves diastolic function in part by preventing cardiac fibrosis. This antifibrotic effect can be at least partially attributed to inhibition of a profibrotic cardiac fibroblast phenotype, likely through Gα_i inhibiting the cAMP/PKA/CREB signaling axis.