## 3 Results
### 7 KCh Affects Transcription of Lipid Metabolism-Related Genes
The principal component analysis (PCA) plot of the mRNA-seq data from control (green) and 7 KCh treatment (red) groups is shown. (**B**) The volcano plot highlights the upregulated (up, red) and downregulated (down, blue) genes in 7 KCh-treated cells (versus Con cells). The fold change (\(\log_{2}\left(\frac{\text{KCh}}{\text{Con}}\right)\)) is indicative of \(\log_{2}\left(\frac{\text{Expression units (in PFKM)}}{\text{Expression units (in PFKM)}}\right)\) of the gene in \(\text{Control}\left(\text{Con}\left(\text{cells}\right)\right)\) for the specified gene (\(n=3\), q value \(<0.001\)). (**C**) Gene association network and module crosstalk network of all significantly altered genes are shown. The default network was generated by Network Analyst. The size of the nodes is based on their degree values, with big size for large degree values. The color of nodes is proportional to their betweenness centrality values. Alteration in Gene Expression and Metabolites Involved in MVA Pathway in 7 KCh-Treated Cardiac Cells  
Of the 7 KCh-induced transcriptomic changes in HL-1 cells, the expression of a number of genes was altered. These genes encode enzymes involved in the MVA pathway and the subsequent conversion reactions, such as _Fdps_, _Hmgsc1_, _Idi1_, _Mvd_, _Hmgcr_, _Acat2_, _Mvk_, _Pmuvk_, _Pcyox1_, _Dhds_, and _Coq2_ (Figure 4A). There were decreases in expression of the MVA pathway-associated genes in 7 KCh-treated cells, compared to those of control (Con) cells (Figure 4B). Downregulation of these genes may affect the biosynthesis of Chol (Figure 4C), dolichol (Figure 4D), and coenzyme Q (Figure 4E). As expected, the levels of MVA derivatives geranyl pyrophosphate (GPP) and farnesyl-pyrophosphate (FPP) decreased in 7 KCh-treated cells, whereas MVA-5-pyrophosphate (M5 PP) and geranylgeranyl pyrophosphate (GGPP) levels remained unchanged (Figure 4C). Downstream products dolichols and CoQs are mildly affected by 7 KCh. The dolichol-18 level was reduced modestly but significantly, while the dolichol-19 level decreased nonsignificantly. CoQ9 did not change, while the CoQ10 level was significantly reduced in the 7 KCh-treated cells (Figure 4D,E). The Chol level did not decrease significantly (Figure 1B,E), while a number of cholesterol esters (CEs) increased in abundance (Figure 5A). Figure 4: _Cont._Figure 4: Effect of 7 KCh on MVA pathway in cardiac cells. (**A**) A simplified diagram illustrating the mevalonate (MVA) pathway and its conversion to cholesterol, dolichol, coenzyme Q, and farnesyl-PP and geranylgeranyl-PP. Genes marked in blue represent the downregulated genes after 7 KCh treatment, while those marked in red represent the upregulated ones. (**B**) Changes in expression levels of MVA pathway-related genes in HL-1 cells treated with 20 \(\mu\)M 7 KCh for 24 h are shown as the fold change (\(\log_{2}\left(\frac{\text{TKCh}}{\text{Con}}\right)\)) that is indicative of \(\log_{2}\left(\frac{\text{Expression units (in FFKM) of the gene in 7 KCh - treated cells}}{\text{Expression units (in FFKM) of the gene in control (Con)cells}}\right)\) for the specified gene. The differentially changed genes include _Hmges1_, _Idi1_, _Hmgcr_, _Mvk_, _Acat2_, _Mvd_, _Pmrk_, _Fdps_, _Pcyox1_, _Coy2_ and _Dh4 ds_ (adjusted \(p\) value \(<0.05\), \(n=3\)). The accession number and descriptions of these genes are given in Supplementary Table S1. (**C-E**) HL-1 cells were untreated (_Control_) or treated with indicated concentrations of 7 KCh for 24 h and harvested for LC-MS/MS-based determination of intermediates in the MVA pathway (**C**), dolichols (**D**), and CoQs (**E**). Data are mean \(\pm\) SD of three experiments. * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\). Mevalonate, MVA; mevalonate-5-pyrophosphate, M5 PP; geranyl pyrophosphate, GPP; famesyl pyrophosphate, FPP; geranylgeranyl pyrophosphate, GGPP; Dolichol, Dol; coenzyme Q, CoQ.