Abstract Body (Do not enter title and authors here): Introduction
Chronic heart failure (HF) is linked to elevated serum TNF-α levels and affects multiple signaling pathways in non-cardiomyocytes, such as immune cells, intestinal epithelial cells, lymphatic endothelial cells, vascular cells, and their interactions. The combined dysbiosis of host transcriptomics and gut microbiota concerning altered TNF-α signaling in older adults with HF remains unknown.

Methods
We recruited 10 older adults with heart failure (HF) (6 females) and 16 healthy controls (HCs) (10 females) from the Northeastern U.S. Non-fasting peripheral blood and stool samples were collected. Serum TNF-α was assayed using Enzyme-linked Immunosorbent Assay (ELISA) kits. Differentially expressed genes (DEGs) between HF and HCs were investigated using the R package "DESeq2" after aligning the raw blood RNA sequence data to the reference database and undergoing quality control. The QIAGEN Ingenuity Pathway Analysis (IPA) was used to analyze the canonical pathways associated with the DEGs. The 16S rRNA V4 gene regions of stool samples were sequenced and processed using the Mothur 1.42.3 pipeline. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to predict the metagenomic functions of different gut microbiota compositions.

Results
The mean ages of the HF and HC subjects were 73.50 (SD = 8.33) and 63.19 (SD = 7.75), respectively. HF subjects had significantly higher serum TNF-α levels than HCs (p < 0.05). Among the DEGs, HF subjects had 18 downregulated genes (e.g., AK5, FAM167A, RGCC, and SARDH) and 3 upregulated genes (SMPD3, TMIGD3, and FRRS1) compared with HCs. TNF signaling (p < 0.01) was one of the significantly different canonical pathways in the DEGs between HF and HCs. HF subjects had significantly enriched Mogibacterium and diminished Sutterella than HCs (p < 0.05) and lower P53 signaling pathway activity than HCs (p < 0.05) among the predicted functions in stool samples.

Conclusions
By analyzing serum TNF-α, whole transcriptomics, and gut microbiota, we identified higher serum TNF-α, differentially expressed genes (DEGs) and their canonical pathways, and distinct compositions and predicted functions of gut microbiota in older adults with HF compared to healthy controls. These findings suggest that TNF-α signaling may be a potential target for developing precise HF interventions and highlight the need for further large-scale multi-omics analysis in understanding and treating HF.