Background: Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored. Methods: C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. Results: Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate. Conclusions: Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate’s preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Fixation of samples is broadly used prior to the histological evaluation of tissue samples. Though recent reports demonstrated the ability to use fixed tissues for mass spectrometry imaging (MSI) based proteomics, glycomics and tumor classification studies, to date comprehensive evaluation of fixation-related effects for spatially resolved metabolomics and drug disposition studies is still missing. In this study we used matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) MSI to investigate the effect of formalin-fixation and formalin-fixation combined with paraffin embedding on the detectable metabolome including xenobiotics. Formalin fixation was found to cause significant washout of polar molecular species, including inorganic salts, amino acids, organic acids and carnitine species, oxidation of endogenous lipids and formation of reaction products between lipids and fixative ingredients. The slow fixation kinetics under ambient conditions resulted in increased lipid hydrolysis in the tissue core, correlating with the time-dependent progression of the fixation. Paraffin embedding resulted in subsequent partial removal of structural lipids resulting in the distortion of the elucidated biodistributions.

Transcriptomic studies have attempted to classify glioblastoma (GB) into subtypes that predict survival and have different therapeutic vulnerabilities^1–3. Here we identified three metabolic subtypes: glycolytic, oxidative and a mix of glycolytic and oxidative, using mass spectrometry imaging of rapidly excised tumour sections from two patients with GB who were infused with [U-¹³C]glucose and from spatial transcriptomic analysis of contiguous sections. The phenotypes are not correlated with microenvironmental features, including proliferation rate, immune cell infiltration and vascularization, are retained when patient-derived cells are grown in vitro or as orthotopically implanted xenografts and are robust to changes in oxygen concentration, demonstrating their cell-intrinsic nature. The spatial extent of the regions occupied by cells displaying these distinct metabolic phenotypes is large enough to be detected using clinically applicable metabolic imaging techniques. A limitation of the study is that it is based on only two patient tumours, albeit on multiple sections, and therefore represents a proof-of-concept study.