Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction. Protocatechuic aldehyde (PCA) is a major phenolic acid in Chinese herb Danshen (Salvia miltiorrhiza root). This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2 (PKM2) function to protect cardiomyocytes. In rats subjected to isoprenaline, PCA attenuated heart injury and protected cardiomyocytes from apoptosis. Through DARTS and CETSA assays, we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation (OGD). In the nucleus, PCA increased the binding of PKM2 to β-catenin via preserving PKM2 acetylation, and the complex, in cooperation with T-cell factor 4 (TCF4), was required for transcriptional induction of genes encoding anti-apoptotic proteins, contributing to rescuing cardiomyocyte survival. In addition, PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2. Consistently, PCA increased the binding of PKM2 to β-catenin, improved heart contractive function, normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation, but the protective effects were lost in Pkm2-deficient heart. Together, we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via β-catenin/TCF4 signaling cascade, suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.

Ischemic preconditioning (IPC) is a potential intervention known to protect the heart against ischemia/reperfusion injury, but its role in the no-reflow phenomenon that follows reperfusion is unclear. Dihydrotanshinone I (DT) is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC. Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation (OGD), but the protection was prevented by a ROS scavenger. In addition, DT administration protected the heart against isoprenaline challenge. Mechanistically, PKM2 reacted to transient ROS via oxidization at Cys423/Cys424, leading to glutathionylation and nuclear translocation in dimer form. In the nucleus, PKM2 served as a co-factor to promote HIF-1α-dependent gene induction, contributing to adaptive responses. In mice subjected to permanent coronary ligation, cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection, which was rescued by overexpression of wild-type Pkm2, rather than Cys423/424-mutated Pkm2. In conclusion, PKM2 is sensitive to oxidation, and subsequent glutathionylation promotes its nuclear translocation. Although IPC has been viewed as a protective means against reperfusion injury, our study reveals its potential role in protection of the heart from no-reflow ischemia.

Amyloid fibril formation is a hallmark of diverse neurodegenerative and metabolic diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and type 2 diabetes mellitus (T2DM). Conventional diagnosis is based on the appearance of fibrils or plaques, while neglects the role of early-stage oligomers in the disease progression. Recent studies have uncovered that it is the early-stage oligomer, rather than the mature fibril, that greatly contributes cytotoxicity. The formation of oligomers involves complicate structural conversions and it is essential to investigate their conformational changes for a better understanding of aggregation mechanism. The coexistence of soluble early-stage oligomers, intermediates, and pre-fibril species makes it difficult to be differentiate by morphological methods, and only average structural information is provided as they lack the ability of separation. Therefore, mass spectrometry (MS) becomes an alternative technique that presents new and complementary insights into the onset of amyloid fibrils. This review highlights the hotspots and important achievements by MS in the field of amyloid formation mechanism, including the direct detection and differentiation of soluble oligomers (native MS), unambiguous identification of interacted sites involved in the onset of aggregation [hydrogen/deuterium exchange (HDX) and chemical cross-linking (CX)], and conformational switch that leads to fibrilization [collision cross section (CCS) regularity by ion mobility (IM)].

Discovery of disease biomarker based on untargeted metabolomics is informative for pathological mechanism studies and facilitates disease early diagnosis. Numerous of metabolomic strategies emerge due to different sample properties or experimental purposes, thus, methodological evaluation before sample analysis is essential and necessary. In this study, sample preparation, data processing procedure and metabolite identification strategy were assessed aiming at the discovery of biomarker of breast cancer. First, metabolite extraction by different solvents, as well as the necessity of vacuum-dried and re-dissolution, was investigated. The extraction efficiency was assessed based on the number of eligible components (components with MS/MS data acquired), which was more reasonable for metabolite identification. In addition, a simplified data processing procedure was proposed involving the OPLS-DA, primary screening for eligible components, and secondary screening with constraints including VIP, fold change and p value. Such procedure ensured that only differential candidates were subjected to data interpretation, which greatly reduced the data volume for database search and improved analysis efficiency. Furthermore, metabolite identification and annotation confidence were enhanced by comprehensive consideration of mass and MS/MS errors, isotope similarity, fragmentation match, and biological source confirmation. On this basis, the optimized strategy was applied for the analysis of serum samples of breast cancer, according to which the discovery of differential metabolites highly encouraged the independent biomarkers/indicators used for disease diagnosis and chemotherapy evaluation clinically. Therefore, the optimized strategy simplified the process of differential metabolite exploration, which laid a foundation for biomarker discovery and studies of disease mechanism.