Rapid fractionation of mitochondria from mouse liver and heart reveals in vivo metabolite compartmentation.

The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 s stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable the assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.

Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury.

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.

Rapid and selective generation of H2S within mitochondria protects against cardiac ischemia-reperfusion injury.

Mitochondria-targeted H2S donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing H2S that decreases oxidative damage. However, the rate of H2S release by current donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases H2S within mitochondria. MitoPerSulf is quickly taken up by mitochondria, where it reacts with endogenous thiols to generate a persulfide intermediate that releases H2S. MitoPerSulf is acutely protective against cardiac IR injury in mice, due to the acute generation of H2S that inhibits respiration at cytochrome c oxidase thereby preventing mitochondrial superoxide production by lowering the membrane potential. Mitochondria-targeted agents that rapidly generate H2S are a new class of therapy for the acute treatment of IR injury.

Mitochondrial metabolism and bioenergetic function in an anoxic isolated adult mouse cardiomyocyte model of in vivo cardiac ischemia-reperfusion injury.

Cell models of cardiac ischemia-reperfusion (IR) injury are essential to facilitate understanding, but current monolayer cell models poorly replicate the in vivo IR injury that occurs within a three-dimensional tissue. Here we show that this is for two reasons: the residual oxygen present in many cellular hypoxia models sustains mitochondrial oxidative phosphorylation; and the loss of lactate from cells into the incubation medium during ischemia enables cells to sustain glycolysis. To overcome these limitations, we incubated isolated adult mouse cardiomyocytes anoxically while inhibiting lactate efflux. These interventions recapitulated key markers of in vivo ischemia, notably the accumulation of succinate and the loss of adenine nucleotides. Upon reoxygenation after anoxia the succinate that had accumulated during anoxia was rapidly oxidized in association with extensive mitochondrial superoxide/hydrogen peroxide production and cell injury, mimicking reperfusion injury. This cell model will enable key aspects of cardiac IR injury to be assessed in vitro.

Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition.

The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling.

Abrogating mitochondrial ROS in neurons or astrocytes reveals cell-specific impact on mouse behaviour.

Cells naturally produce mitochondrial reactive oxygen species (mROS), but the in vivo pathophysiological significance has long remained controversial. Within the brain, astrocyte-derived mROS physiologically regulate behaviour and are produced at one order of magnitude faster than in neurons. However, whether neuronal mROS abundance differentially impacts on behaviour is unknown. To address this, we engineered genetically modified mice to down modulate mROS levels in neurons in vivo. Whilst no alterations in motor coordination were observed by down modulating mROS in neurons under healthy conditions, it prevented the motor discoordination caused by the pro-oxidant neurotoxin, 3-nitropropionic acid (3-NP). In contrast, abrogation of mROS in astrocytes showed no beneficial effect against the 3-NP insult. These data indicate that the impact of modifying mROS production on mouse behaviour critically depends on the specific cell-type where they are generated.

Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly.

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide-dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2 In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.