## 3 Results
### 7 KCh Affects Transcription of Lipid Metabolism-Related Genes  
The PCA plot revealed significant changes in transcriptomes of control and 7 KCh-treated cells (Figure 3A). Expression of 401 genes changed significantly in 7 KCh-treated HL-1 cells versus untreated control (adjusted \(p\)-value < 0.01 and fold change > 1.5). These genes included 257 upregulated and 144 downregulated genes. These differentially expressed genes (DEGs) are presented by a volcano plot (Figure 3B). Data mapping to KEGG and Reactome revealed several significantly altered pathway gene sets. These pathway gene sets included those implicated in Chol biosynthesis, sterol regulatory element-binding transcription factor (SREBF)-mediated gene activation, SREBP-regulated Chol biosynthesis, metabolism of lipids and lipoproteins, and ATF4-mediated gene activation (Figure 3C). These enriched pathways with differentially upregulated and downregulated genes are summarized in Table 1. The gene sets associated with ATF4-mediated gene activation, PERK regulated-pathway, and unfolded protein response pathway were upregulated (Figure 3C and Table 1) after 24 h-treatment of HL-1 cells with 20 \(\upmu\)M 7 KCh. \begin{table}
\begin{tabular}{l c c c c c} \hline
**Pathway** & **Total** & **Expected** & **Hits** & \(p\) **Value** & **FDR** \\ \hline \hline \multicolumn{6}{l}{UPEguated genes in 7 KCh-treated cardiac cell} \\ Activation of Genes by ATF4 & 7 & 0.047 & 4 & \(6.12\times 10^{-8}\) & \(8.02\times 10^{-5}\) \\ PERK regulated gene expression & 10 & 0.0671 & 4 & \(3.62\times 10^{-7}\) & \(2.37\times 10^{-4}\) \\ Unfolded Protein Response & 66 & 0.443 & 6 & \(4.38\times 10^{-6}\) & \(1.91\times 10^{-3}\) \\ Circadian Clock & 39 & 0.262 & 4 & \(0.000123\) & \(3.22\times 10^{-2}\) \\ PPARA Activates Gene Expression & 78 & 0.523 & 5 & \(0.00016\) & \(3.22\times 10^{-2}\) \\ Amino acid synthesis and interconversion (transamination) & 21 & 0.141 & 3 & \(0.000346\) & \(3.48\times 10^{-2}\) \\ Downregulated genes in 7 KCh-treated cardiac cell & & & & & \\ Cholesterol biosynthesis & 28 & 0.449 & 18 & \(1.35\times 10^{-26}\) & \(1.77\times 10^{-23}\) \\ Activation of Gene Expression by SREBP (SREBF) & 32 & 0.513 & 15 & \(2.04\times 10^{-19}\) & \(1.33\times 10^{-16}\) \\ Regulation of Cholesterol Biosynthesis by SREBP (SREBF) & 49 & 0.786 & 16 & \(1.36\times 10^{-17}\) & \(5.94\times 10^{-15}\) \\ Metabolism of lipids and lipoproteins & 553 & 8.87 & 37 & \(1.08\times 10^{-14}\) & \(3.54\times 10^{-12}\) \\ Metabolism & 1600 & 25.6 & 55 & \(7.37\times 10^{-10}\) & \(1.93\times 10^{-7}\) \\ Fatty Acyl-CoA Biosynthesis & 18 & 0.289 & 6 & \(2.36\times 10^{-7}\) & \(5.16\times 10^{-5}\) \\ Collagen biosynthesis and modifying enzymes & 52 & 0.834 & 8 & \(1.42\times 10^{-6}\) & \(2.56\times 10^{-4}\) \\ Triglyceride Biosynthesis & 37 & 0.594 & 7 & \(1.57\times 10^{-6}\) & \(2.56\times 10^{-4}\) \\ Extracellular matrix organization & 159 & 2.55 & 12 & \(7.90\times 10^{-6}\) & \(1.00\times 10^{-3}\) \\ Assembly of collagen fibrils and other multimeric structures & 47 & 0.754 & 7 & \(8.38\times 10^{-6}\) & \(1.00\times 10^{-3}\) \\ Fatty acid, triacylglycerglycerol, and ketone body metabolism & 160 & 2.57 & 12 & \(8.43\times 10^{-6}\) & \(1.00\times 10^{-3}\) \\ Collagen formation & 73 & 1.17 & 8 & \(1.93\times 10^{-5}\) & \(2.10\times 10^{-3}\) \\ Degradation of collagen & 54 & 0.867 & 7 & \(2.15\times 10^{-5}\) & \(2.17\times 10^{-3}\) \\ Integrin cell surface interactions & 86 & 1.38 & 8 & \(6.39\times 10^{-5}\) & \(5.98\times 10^{-3}\) \\ Degradation of the extracellular matrix & 87 & 1.4 & 8 & \(6.95\times 10^{-5}\) & \(6.07\times 10^{-3}\) \\ Ethanol oxidation & 8 & 0.128 & 3 & \(0.000212\) & \(1.74\times 10^{-2}\) \\ \hline \end{tabular}
\end{table} 
Table 1: Pathways with differentially upregulated and downregulated genes. Figure 3: mRNA-seq profiling of 7 KCh-treated cardiac cells. (**A**) HL-1 cells were untreated (Con) or treated with 20 uM 7 KCh for 24 h and harvested for mRNA-seq analysis by Novogene.