Background: Myocardial ischemia/reperfusion injury is associated with adverse cardiovascular outcomes after acute myocardial infarction. However, the molecular mechanism of ischemia/reperfusion injury remains unclear. Mitochondria dysfunction is a participant in and regulator of myocardial ischemia-reperfusion injury. However, the molecular mechanisms involved in this process are not yet fully understood. We previously reported that Notch1 can reduce mitochondrial lysis, reduce myocardial infarct size, and inhibit ventricular remodeling. Herein, we explore the role of the downstream target Notch1 in mitochondrial regulation. Methods: This study constructs an ischemic/reperfusion injury rat model and a hypoxia/reoxygenation cell model. The expression of PTEN is detected by real-time PCR, Western blot, and immunofluorescence staining. Cell viability is analyzed with CCK-8. Apoptosis level is detected via the TUNEL assay, and mitochondrial fission/fusion is analyzed with MitoTracker Green staining. Cardiac troponin I (cTnI), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and CK levels of creatine kinase-MB (CK) are measured with ELISA kits. Results: We found that PETN-Pink1-Parkin signaling is inhibited by Notch1 I/R in injured neonatal cardiomyocytes and hearts, i.e., via the inhibition of mitochondrial dysfunction and fragmentation. With the recure of PTEN or Pink1, the protective effect of Notch1 was largely diminished. Conclusion: These results suggest that N1ICD acts protectively against ischemic reperfusion injury by suppressing PTEN-Pink1-mediated mitochondrial dysfunction and fragmentation.

Myocarditis is a complex disease characterized by myocardial inflammatory cell infiltration. The purpose of our study was to investigate the gene and single-cell signature to explore the involvement of immune cells in myocarditis. Gene expressions merged from GSE35182 and GSE35182 datasets were subjected to differential expression gene (DEG) analysis and PPI network construction. The correlation analysis of DEGs with immune cell infiltration was performed. Single-cell RNA sequencing (scRNA-seq) was downloaded from GSE174458. A total of 58 DEGs were identified, including 51 DEGs upregulated and 7 DEGs downregulated in the myocarditis group compared with the control group. GO and KEGG enrichment analyses revealed that myocarditis triggered DEGs mainly involved in immune-related processes and pathways. PPI network analysis identified 20 central hub genes. Occurrence of myocarditis induced significant enrichment of conventional dendritic cell 2 (cDC2), plasmacytoid DC, and plasma cell in myocardial tissue. Mmp12, Gpnmb, and Atp6v0d2 expressions were positively correlated with cDC abundance, of which only Mmp1 and Gpnmb were shared with hub gene list. A total of 20972 cells in scRNA-seq yielded 26 cell clusters and annotated 9 cell types, including fibroblasts, neutrophils, stromal cells, monocytes, basophils, B cells, natural killer T cells, innate lymphoid cells, and T cells, and only proportion of natural killer T cells and monocytes were higher in the myocarditis than in control. Monocytes annotated 3 subclusters including DC, macrophage, and monocytes. Hub genes of Ctss, Mpeg1, Cybb, H2-Ab1, Ly86, CD74, and Lgals3 were highly expressed in monocytes cluster. Among DC-correlated DEGs, Mmp12 was mainly expressed in monocyte cluster, and Gpnmb was mainly expressed in fibroblast cluster, whereas Atp6v0d2 expression has a weaker signal and weaker cell preference. In conclusion, DC infiltration and its associated pivotal genes may be responsible for progression of myocarditis. Our study expands and provides novel information on the immune cell engagement of myocarditis.