Gpx3 and Egr1 Are Involved in Regulating the Differentiation Fate of Cardiac Fibroblasts under Pressure Overload.

Objectives: Although myocardial fibrosis is a common pathophysiological process associated with many heart diseases, the molecular mechanisms regulating the development of fibrosis have not been fully determined. Recently, single cell RNA sequencing (scRNA-seq) analysis has been used to examine cellular fate and function during cellular differentiation and has contributed to elucidating the mechanisms of various diseases. The main purpose of this study was to characterize the fate of cardiac fibroblasts (CFs) and the dynamic gene expression patterns in a model of cardiac pressure overload using scRNA-seq analysis. Methods: The public scRNA-seq dataset of the transverse aortic coarctation (TAC) model in mice was downloaded from the GEO database, GSE155882. First, we performed quality control, dimensionality reduction, clustering, and annotation of the data through the Seurat R package (v4.0.5). Then, we constructed the pseudotime trajectory of cell development and identified key regulatory genes using the Monocle R package (v2.22.0). Different cell fates and groups were fully characterized by Gene Set Enrichment Analysis (GSEA) analysis and Transcription factor (TF) activity analysis. Finally, we used Cytoscape (3.9.1) to extensively examine the gene regulatory network related to cell fate. Results: Pseudotime analysis showed that CFs differentiated into two distinct cell fates, one of which produced activated myofibroblasts, and the other which produced protective cells that were associated with reduced fibrosis levels, increased antioxidative stress responses, and the ability to promote angiogenesis. In the TAC model, activated CFs were significantly upregulated, while protective cells were downregulated. Treatment with the bromodomain inhibitor JQ1 reversed this change and improved fibrosis. Analysis of dynamic gene expression revealed that Gpx3 was significantly upregulated during cell differentiation into protective cells. Gpx3 expression was affected by JQ1 treatment. Furthermore, Gpx3 expression levels were negatively correlated with the different levels of fibrosis observed in the various treatment groups. Finally, we found that transcription factors Jun, Fos, Atf3, and Egr1 were upregulated in protective cells, especially Egr1 was predicted to be involved in the regulation of genes related to antioxidant stress and angiogenesis, suggesting a role in promoting differentiation into this cell phenotype. Conclusions: The scRNA-seq analysis was used to characterize the dynamic changes associated with fibroblast differentiation and identified Gpx3 as a factor that might be involved in the regulation of myocardial fibrosis under cardiac pressure overload. These findings will help to further understanding of the mechanism of fibrosis and provide potential intervention targets.

Inhibition of autophagy by geniposide protects against myocardial ischemia/reperfusion injury.

Geniposide (GP), extracted from a traditional Chinese herb Gardenia jasminoides, has extensive pharmacological effects. But the effects and the potential mechanisms of GP on myocardial ischemia/reperfusion (I/R) injury are poorly understood. In present study, we investigated the effect of GP on myocardial I/R injury in vivo and hypoxia/reoxygenation (H/R) in vitro respectively, and its mechanism. The results showed that GP reduced myocardial infarct size, alleviated acute myocardial injury, improved cardiac function, regulated apoptosis-related proteins and inhibited apoptosis. In vitro experiments revealed that GP enhanced the cell viability, regulated apoptosis-related proteins and prevented cell apoptosis during H/R in H9c2 cells. GP inhibited the expression of autophagy-related proteins and autophagosome accumulation both in vivo and in vitro. The effects of GP were blocked by rapamycin (RAPA) administration. In summary, our results showed that GP protected against myocardial I/R injury and involved inhibition of autophagy, which might be through activating AKT/mTOR signaling pathways.

Trimetazidine protects against myocardial ischemia/reperfusion injury by inhibiting excessive autophagy.

Trimetazidine (TMZ) has been demonstrated to have protective effects against myocardial ischemia/reperfusion (MI/R) injury. In the present study, we investigated the effects and the underlying mechanisms of TMZ on autophagy during MI/R in vivo and in vitro. In the in vivo study, an animal model of MI/R was induced by coronary occlusion. TMZ (20 mg/kg/day) protected the rat hearts from MI/R-induced heart failure by increasing ejection fraction and fractional shortening and decreasing end-systolic volume, end-diastolic volume, left ventricular (LV) internal diameter at systole, and LV internal diameter at diastole; it alleviated myocardial injury and oxidative stress by decreasing LDH, creatine kinase MB isoenzyme, ROS, and MDA levels and increasing SOD and glutathione peroxidase levels in plasma. TMZ also reduced myocardial infarct size and apoptosis. Moreover, TMZ markedly inhibited MI/R-induced autophagy by decreasing the protein and messenger RNA levels of LC3-II, Beclin1, ATG5, and ATG7 and the number of autophagosomes and by involving the AKT/mTOR pathway. Further, in the in vitro experiments, H9c2 cells were incubated with TMZ (40 µM) to explore the direct effects of TMZ following exposure to hypoxia and reoxygenation (H/R). TMZ increased cell viability and the concentration of intracellular SOD and inhibited H/R-induced cell apoptosis and ROS production. Moreover, TMZ decreased the number of autophagosomes and autophagy-related protein expression; it also upregulated p-AKT and p-mTOR expression. In addition, TMZ augmented Bcl-2 protein expression and diminished Bax protein expression, the Bax/Bcl-2 rate, and cleaved caspase-3 level. However, these effects on H9c2 cells were notably abolished by the PI3K inhibitor LY294002. In conclusion, our results showed that TMZ inhibited I/R-induced excessive autophagy and apoptosis, which was, at least partly, mediated by activating the AKT/mTOR pathway. KEY MESSAGES: TMZ improved cardiac function, alleviated myocardial injury and oxidative stress, and reduced the myocardial infarct area and apoptosis. TMZ inhibited MI/R-induced myocardial autophagy, H/R-induced H9c2 cell apoptosis, and autophagy flux. The effect of TMZ on autophagy was repressed by LY294002. TMZ protected against MI/R injury by inhibiting excessive autophagy via activating the AKT/mTOR pathway.