## 3 Results
### The Metabolic Profiles of 7 KCh-Treated HL-1 Cells  
To study the changes in global metabolism of cardiomyocytes in response to 7 KCh, we treated HL-1 cells without (i.e., vehicle-treated) or with different concentrations of 7 KCh, and then analyzed the metabolome using LC-TOF-MS. Typical spectra of HL-1 cell extract were obtained in positive and negative ion modes. After data processing with Progenesis QI, the data were analyzed with SIMCA-P. The OPLS-DA score plots are shown in Figure 2. The score plots showed that the metabolite profiles of HL-1 cells treated with different 7 KCh concentrations were spatially separated in both ESI positive and negative modes (Figure 2A,B). The metabolite profiles of HL-1 changed significantly in a dose-dependent manner after 7 KCh treatment. Fifty-four and 127 metabolites acquired in ESI positive and negative modes were selected based on the criteria of variable importance in the projection (VIP) scores > 3.0 and significant differences (_p_ < 0.01) between the untreated and those treated with 20 uM 7 KCh (Figure 2C,D). The top 50 significantly changed metabolites are presented in Figure 2E,F. These findings suggest that sterols, phospholipids, and lysophospholipids are important discriminators of untreated cells and those treated with 7 KCh. The levels of lysophospholipids (such as lysophosphatidylcholines (lysoPCs) and lysophosphatidylethanolamines (lysoPEs)), and oxysterols were elevated, while the levels of many phospholipids decreased in 7 KCh-treated cells compared with those of the untreated cells. Furthermore, glutathione decreased in 7 KCh-treated cells.  
Figure 1: Growth inhibition and 7 KCh accumulation in HL-1 cells. (**A–C**) HL-1 cells (\(5\times 10^{4}\)/well) were treated without (Con) or with 20 uM 7 KCh (or Chol) for indicated periods (0, 1, 3, 6, 12, and 24 h). These cells were harvested for LC-MS-based quantification of the intracellular 7 KCh (**A**) and Chol (**B**). Data are mean \(\pm\) SD of three experiments. The numbers of the cells treated for different periods were determined and expressed as the percentage of that of the untreated HL-1 cells (**C**). Data are mean \(\pm\) SD of three experiments. * \(p<0.05\), ** \(p<0.01\), *** \(p<0.005\), vs. Con. (**D–F**) Cells were treated with different concentrations (0, 10, and 20 uM) of 7 KCh for 24 h and then harvested for quantification of intracellular 7 KCh (**D**) and Chol (**E**). Data are mean \(\pm\) SD of six experiments. *** \(p<0.005\), vs. Con. The numbers of the cells treated with different 7 KCh concentrations were determined and are expressed as the percentage of that of untreated HL-1 cells (**F**). Data are mean \(\pm\) SD of six experiments. *** \(p<0.005\), vs. Con.  
Figure 2: Changes in global metabolism in 7 KCh-treated HL-1 cells. For global metabolomic analysis, HL-1 cells were untreated (Con) or treated with the indicated concentrations (10, 20 uM) of 7 KCh for 24 h, and harvested for subsequent analyses by LC-MS and LC-MS/MS in ESI-positive (**A**,**C**,**E**) and ESI-negative (**B**,**D**,**F**) modes. The corresponding OPLS-DA score plots are shown (**A**,**B**). Significantly different metabolites between 7 KCh-treated and control cells were selected with VIP score > 3 and \(p\) value < 0.01. The log\({}_{2}\) fold changes in the abundance of metabolites differentially abundant in cells treated with 20 uM 7 KCh versus Con cells (i.e., the log\({}_{2}\) of the ratio of the abundance of a metabolite in 7 KCh group to that of Con group) are plotted (**E**,**F**). The lysophospholipids are highlighted by the red bars.