Abstract Body (Do not enter title and authors here): Background: Heart failure (HF) is a complex syndrome marked by adverse cardiac remodeling and natriuretic peptide (NP) deficiency, which drives disease progression despite current therapies. NPs exert cardioprotective effects via particulate guanylate cyclase receptors GC-A and GC-B, which generate cGMP. Activation of GC-A by ANP or BNP induces arteriodilation, natriuresis, antihypertrophic effects, and inhibits the renin-angiotensin-aldosterone system, while GC-B activation by CNP mediates antifibrotic, lusitropic, venodilatory, and anti-inflammatory actions. Existing drugs target either GC-A or GC-B, but none target both. We developed RT-A1, a unimolecular peptide activating both GC-A and GC-B, hypothesizing it will attenuate myocardial remodeling in a preclinical model of HF with preserved systolic function.
Methods: RT-A1 was engineered by rational drug design. HEK293 cells expressing human GC-A or GC-B, human cardiac myocytes (CMs) and fibroblasts (CFs) were treated with RT-A1 (10-100nM) and cGMP was measured. In vivo, mice underwent mild transverse aortic constriction (TAC) or sham surgery. After 3 weeks, LV hypertrophy (LVH) was confirmed by echo. TAC mice were then randomized to vehicle (Veh) or RT-A1 (50pmol/kg/min) via subcutaneous osmotic pumps for 3 weeks. Endpoints included LV/body weight ratio, myocyte cross-sectional area (CSA), plasma cGMP, and RNA-seq. Data are mean±SEM. P<0.05 was significant.

Results: RT-A1 dose-dependently increased cGMP in HEK293 cells expressing GC-A or GC-B, confirming dual activation, and elevated cGMP in human CMs and CFs. TAC induced significant LV wall thickening with preserved systolic function versus sham. After three weeks, RT-A1 normalized LV/body weight (3.14±0.14 vs. 3.82±0.29, P=0.03) and reduced myocyte CSA (348±16 vs. 506±17, P=0.0005) compared to Veh. Plasma cGMP was higher in TAC+RT-A1 versus TAC+Veh (85±8 vs 54±8 pmol/ml, P=0.01), confirming target engagement. RNA-seq showed reduced LV BNP expression and downregulation of hypertrophy, fibrosis, and inflammation pathways with RT-A1 treatment vs. Veh following TAC.

Conclusions: RT-A1 is a novel dual GC-A/GC-B peptide activator that elevates plasma cGMP and reduces adverse cardiac remodeling in experimental HF by downregulating hypertrophy, fibrosis, and inflammation pathways. Dual GC-A/GC-B activation via RT-A1 represents a promising anti-remodeling therapeutic strategy for HF with preserved systolic function warranting further study.