Mechanistic investigation of Guizhi Gancao decoction in alleviating isoproterenol-induced chronic heart failure via inhibition of sarcoplasmic/endoplasmic (SR/ER) reticulum Ca2+-ATPase 2a ubiquitination

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic heart failure (CHF) remains a leading cause of mortality globally, with limited therapeutic options targeting its underlying pathological mechanisms. Guizhi Gancao Decoction (GGD), a classic traditional Chinese medicine formula, has been used for centuries to treat cardiovascular diseases, but its molecular mechanisms in CHF remain elusive. AIM OF THE STUDY: This study aimed to investigate the therapeutic effects of GGD on isoproterenol (ISO)-induced CHF in mice and explore its underlying mechanisms, focusing on the cAMP/PKA signaling pathway, ubiquitin-proteasome system (UPS), and calcium handling. METHODS: A CHF model was established by subcutaneous injection of ISO in C57BL/6 mice. Cardiac function and structural remodeling were evaluated using echocardiography, histopathology (hematoxylin-eosin staining, Masson staining), and molecular markers (ANP, BNP). Serum metabolomics combined with western blotting was used to identify key signaling pathways. Co-immunoprecipitation (Co-IP) and mass spectrometry (MS) were employed to explore protein-protein interactions. Molecular docking and in vitro calcium transient assays validated the functional relevance of key targets. RESULTS: GGD treatment significantly improved ISO-induced cardiac dysfunction, as evidenced by increased ejection fraction (EF), fractional shortening (FS), and reduced left ventricular internal diameters (LVIDs/d). Pathological hypertrophy and fibrosis were attenuated by GGD, particularly at low doses, with downregulated ANP/BNP expression. Metabolomic profiling identified the cAMP/PKA pathway as a critical target, where GGD normalized ISO-induced upregulation of PKA regulatory (RI-α/β) and catalytic (α/β/γ) subunits. Five active ingredients in GGD (e.g., 4-hydroxy cinnamic acid, 18α-glycyrrhetinic acid) exhibited strong binding affinity to PKA. Co-IP-MS revealed PKA interaction with proteasome-related proteins (Psma2, Psmb5, Ube2d3), and GGD inhibited ISO-enhanced PKA-proteasome binding, restoring UPS homeostasis. Importantly, GGD reduced ubiquitin-mediated degradation of SERCA2a, preserved calcium transient dynamics, and improved cardiomyocyte contractility, an effect mimicked by the proteasome inhibitor MG132. CONCLUSION: GGD alleviates ISO-induced CHF by modulating the cAMP/PKA pathway, inhibiting PKA-proteasome interactions, and preserving SERCA2a-dependent calcium cycling. These findings highlight GGD as a promising therapeutic agent for CHF via targeting UPS-mediated SERCA2a degradation. However, because the present study was conducted using a single batch of GGD, the findings should be considered preliminary and require further validation across multiple batches of herbal materials.