Abstract Body (Do not enter title and authors here): Introduction
The role of β-adrenergic receptor (βAR) signaling in heart failure with preserved ejection fraction (HFpEF) remains poorly understood. While β-blockers are effective in heart failure with reduced ejection fraction (HFrEF), they offer minimal benefit in HFpEF, suggesting disease-specific differences in adrenergic signaling. This study aims to investigate and compare subcellular adrenergic signaling in adult ventricular myocytes (AVMs) from mouse models of HFpEF and HFrEF.

Methods
We analyzed RNA-seq data to assess βAR and downstream signaling gene expression in human HFpEF and HFrEF samples. To examine subcellular protein kinase A (PKA) activity, we developed FRET-based biosensors targeted to nanodomains at ryanodine receptor 2 (RyR2) and sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a). We evaluated local adrenergic-PKA signaling in AVMs isolated from HFpEF mice (induced by high-fat diet with L-NAME) and HFrEF mice (induced by chronic isoproterenol infusion). We further assessed calcium handling, contractility, and the effects of phosphodiesterase 4D (PDE4D) inhibition.

Results
RNA-seq and Western blot analysis showed β1AR downregulation in human HFrEF and the isoproterenol-induced mouse HFrEF model (n=10), resulting in selective impairment of adrenergic-PKA signaling at RyR2 nanodomains. In contrast, β1AR expression remained unchanged in human HFpEF and HFD+L-NAME mouse hearts. However, PDE4D isoforms were upregulated and selectively associated with SERCA2a, leading to suppression of local adrenergic-PKA signaling (n=10). PDE4D inhibition rescued PKA activity specifically at the SERCA2a nanodomain, enhanced phospholamban phosphorylation, restored calcium cycling, and improved contractile function in HFpEF AVMs. In vivo, treatment with a PDE4 inhibitor ameliorated diastolic dysfunction in HFD+L-NAME mice (n=10).

Conclusions
This study demonstrates distinct subcellular βAR-PKA signaling alterations in HFpEF versus HFrEF. While HFrEF is characterized by β1AR downregulation and RyR2 domain dysfunction, HFpEF shows preserved β1AR expression but increased PDE4D activity that impairs SERCA2a regulation. These findings identify PDE4D as a novel therapeutic target to restore localized adrenergic signaling and SERCA2a function in HFpEF.