Zhigancao decoction ameliorates ischemic heart failure-related ventricular arrhythmias through attenuation of transforming growth factor-β/Smad-associated myocardial fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhigancao Decoction (ZGCT), also known as Fumai Decoction, is a classical traditional Chinese medicine formula recorded in Shanghan Lun and has long been used for the treatment of "palpitations" and "irregular pulse," corresponding to arrhythmias in modern medicine. Although ZGCT has shown beneficial effects against cardiac arrhythmias, its absorbed bioactive constituents and the underlying mechanisms involved in ischemic heart failure (IHF)-related ventricular arrhythmias (VAs) remain unclear. AIM OF THE STUDY: This study aimed to characterize the chemical profile of ZGCT and identify its circulating and cardiac constituents using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), to identify key pathways through transcriptomic analysis, and evaluate the effects of ZGCT on IHF-related VAs in rats while exploring the potential involvement of myocardial fibrosis and TGF-β/Smad signaling. MATERIALS AND METHODS: UPLC-Q-TOF-MS was employed to characterize the chemical constituents of ZGCT extract and to identify its absorbed components in rat serum and myocardial tissue after administration, thereby defining its in vivo bioactive basis. RNA sequencing (RNA-seq)-based transcriptomic profiling was then performed on myocardial tissues from the control, model, and ZGCT-treated groups to identify differentially expressed genes, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to screen candidate signaling pathways underlying ZGCT activity. In addition, a rat model of IHF was established by permanent ligation of the left anterior descending coronary artery. Rats were randomly assigned to the control, model, low-, medium-, and high-dose ZGCT, metoprolol, SB-431542(a selective TGF-β receptor type I inhibitor), and SB-431542 plus ZGCT groups. Cardiac function was evaluated by echocardiography; ventricular arrhythmia susceptibility, conduction velocity, and electrical conduction dispersion were assessed by burst pacing and multichannel electrical mapping; infarct size and myocardial fibrosis were determined by 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin (H&E), and Masson's trichrome staining; and the expression of TGF-β1, phosphorylated Smad3 (p-Smad3), Smad7, and matrix metalloproteinase 2 (MMP2) was measured by Western blotting and RT-qPCR. RESULTS: UPLC-Q-TOF-MS identified 113 constituents in the ZGCT extract in vitro, of which 80 were detected in serum and 53 were distributed in myocardial tissue after administration. Transcriptomic analysis revealed that ZGCT predominantly regulated the extracellular matrix (ECM)-receptor interaction and TGF-β signaling pathways, with the TGF-β pathway emerging as a putative key target. In IHF rats, ZGCT significantly improved cardiac function and reduced both the incidence and duration of ventricular arrhythmias. Multichannel electrical mapping further demonstrated that ZGCT reversed IHF-induced slowing of conduction velocity and increased conduction dispersion. Mechanistically, ZGCT downregulated the expression of TGF-β1, p-Smad3, and MMP2, while upregulating Smad7. SB-431542 elicited anti-fibrotic and pathway-related molecular changes comparable to those of ZGCT, further supporting a role for TGF-β/Smad signaling in the anti-fibrotic effects of ZGCT. CONCLUSIONS: ZGCT reduced ventricular arrhythmia susceptibility and improved conduction abnormalities in IHF rats in association with attenuation of myocardial fibrosis and modulation of transforming growth factor-β/Smad-related signaling. However, these findings were obtained from a single batch of ZGCT, and further multi-batch validation is required to confirm its chemical consistency and pharmacological reproducibility.