Biology subjects

Milliken, A. S.

Publications and source records attributed to Milliken, A. S..

4 recordsLinked to original sources

bioRxiv2022

Distinct effects of intracellular vs. extracellular acidic pH on the cardiac metabolome during ischemia and reperfusion.

Tissue ischemia results in intracellular pH (pHIN) acidification, and while accumulation of metabolites such as lactate is a known driver of acidic pHIN, less is known about how acidic pHIN regulates metabolism. Furthermore, acidic extracellular (pHEX) during early reperfusion confers cardioprotection, but how this impacts metabolism is unclear. Herein we employed LCMS based targeted metabolomics to analyze perfused mouse hearts exposed to: (i) control perfusion, (ii) hypoxia, (iii) ischemia, (iv) enforced acidic pHIN, (v) control reperfusion, and (vi) acidic pHEX (6.8) reperfusion. Surprisingly little overlap was seen between metabolic changes induced by hypoxia, ischemia, and acidic pHIN. Acidic pHIN elevated metabolites in the top half of glycolysis, and enhanced glutathione redox state. Acidic pHEX reperfusion induced substantial metabolic changes in addition to those seen in control reperfusion. This included elevated metabolites in the top half of glycolysis, prevention of purine nucleotide loss, and an enhancement in glutathione redox state. These data led to parallel hypotheses regarding potential roles for methylglyoxal inhibiting the mitochondrial permeability transition pore, and for acidic inhibition of ecto-5-nucleotidase, as potential mediators of cardioprotection by acidic pHEX reperfusion. However, neither hypothesis was supported by subsequent experiments. In contrast, analysis of cardiac effluents revealed complex effects of pHEX on metabolite transport, suggesting that mildly acidic pHEX may protect in part by enhancing succinate release during reperfusion. Overall, each intervention had distinct and overlapping metabolic effects, suggesting acidic pH is an independent metabolic regulator regardless which side of the cell membrane it is imposed. HIGHLIGHTSO_LIHypoxia, ischemia and acidic pHIN each induce unique cardiac metabolic profiles. C_LIO_LIAcidic pHEX at reperfusion prevents purine loss and enhances succinate release. C_LI

biochemistry↗

bioRxiv2022

Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation

The metabolite succinate accumulates during cardiac ischemia. Within 5 min. of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II (Cx-II). The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking monocarboxylate transporter 1 (MCT-1) may be beneficial in IR, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT-1 would retain succinate in cells, exacerbating ROS generation and IR injury. Using the mitochondrial ROS probe mitoSOX, and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in-situ during the first moments of reperfusion, and found that acute MCT-1 inhibition enhanced mitochondrial ROS generation at reperfusion, and worsened IR injury (recovery of function and infarct size). Both these effects were abrogated by tandem inhibition of Cx-II, suggesting that succinate retention worsens IR due to driving more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator TMRE, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT-1 inhibition. Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation, as key determinants of PT pore opening and IR injury outcomes.

biochemistry↗

bioRxiv2020

Complex effects of pH on ROS from mitochondrial complex II driven complex I reverse electron transport challenge its role in tissue reperfusion injury

Generation of mitochondrial reactive oxygen species (ROS) is an important process in triggering cellular necrosis and tissue infarction during ischemia-reperfusion (IR) injury. Ischemia results in accumulation of the metabolite succinate. Rapid oxidation of this succinate by mitochondrial complex II (Cx-II) during reperfusion reduces the co-enzyme Q (Co-Q) pool, thereby driving electrons backward into complex-I (Cx-I), a process known as reverse electron transport (RET), which is thought to be a major source of ROS. During ischemia, enhanced glycolysis results in an acidic cellular pH at the onset of reperfusion. While the process of RET within Cx-I is known to be enhanced by a high mitochondrial trans-membrane {Delta}pH, the impact of pH itself on the integrated process of Cx-II to Cx-I RET has not been fully studied. Using isolated mitochondria under conditions which mimic the onset of reperfusion (i.e., high [ADP]). We show that mitochondrial respiration (state 2 and state 3) as well as isolated Cx-II activity are impaired at acidic pH, whereas the overall generation of ROS by Cx-II to Cx-I RET was insensitive to pH. Together these data indicate that the acceleration of Cx-I RET ROS by {Delta}pH appears to be cancelled out by the impact of pH on the source of electrons, i.e. Cx-II. Implications for the role of Cx-II to Cx-I RET derived ROS in IR injury are discussed.

biochemistry↗

bioRxiv2019

Controlling the Mitochondrial Protonmotive Force with Light to Impact Cellular Stress Resistance

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called the protonmotive force (PMF) to drive diverse functions and make ATP. Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, reversible method to increase the PMF. To address this issue, we used an optogenetic approach and engineered a mitochondria-targeted light-activated proton pumping protein we called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling outcomes.

physiology↗