Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria‐targeted light‐activated proton pump that we name mitochondria‐ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose‐dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy‐sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well‐characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.

BACKGROUND: Currently no ideal alternative exists for heparin for cardiopulmonary bypass (CPB). Dabigatran is a direct thrombin inhibitor for which a reversal agent exists. The primary end point of the study was to explore whether Dabigatran was an effective anticoagulant for 120 minutes of simulated CPB. METHODS: The study was designed in 2 sequential steps. Throughout, human blood from healthy donors was used for each experimental step. Initially, increasing concentrations of Dabigatran were added to aliquots of fresh whole blood, and the anticoagulant effect measured using kaolin/tissue factor–activated thromboelastography (rapidTEG). The dynamics of all thromboelastography (TEG) measurements were studied with repeated measures analysis of variance (ANOVA). Based on these data, aliquots of blood were treated with high-concentration Dabigatran and placed in a Chandler loop as a simple ex vivo bypass model to assess whether Dabigatran had sufficient anticoagulant effects to maintain blood fluidity for 2 hours of continuous contact with the artificial surface of the PVC tubing. Idarucizumab, humanized monoclonal antibody fragment, was used to verify the reversibility of Dabigatran effects. Finally, 3 doses of Dabigatran were tested in a simulated CPB setup using a heart–lung machine and a commercially available bypass circuit with an arteriovenous (A-V) loop. The primary outcome was the successful completion of 120 minutes of simulated CPB with dabigatran anticoagulation, defined as lack of visible thrombus. Thromboelastographic reaction (R) time was measured repeatedly in each bypass simulation, and the circuits were continuously observed for clot. Scanning Electron Microscopy (SEM) was used to visualize fibrin formation in the filters meshes during CPB. RESULTS: In in vitro blood samples, Dabigatran prolonged R time and reduced the dynamics of clot propagation (as measured by speed of clot formation [Angle], maximum rate of thrombus generation [MRTG], and time to maximum rate of thrombus generation [TMRTG]) in a dose-dependent manner. In the Chandler Loop, high doses of Dabigatran prevented clot formation for 120 minutes, but only at doses higher than expected. Idarucizumab decreased R time and reversed anticoagulation in both in vitro and Chandler Loops settings. In the A-V loop bypass simulation, Dabigatran prevented gross thrombus generation for 120 minutes of simulated CPB. CONCLUSIONS: Using sequential experimental approaches, we showed that direct thrombin inhibitor Dabigatran in high doses maintained anticoagulation of blood for simulated CPB. Idarucizumab reduced time for clot formation reversing the anticoagulation action of Dabigatran.

Mitochondria are organelles that provide energy to cells. Matching energy supply with energy demand is coordinated through various processes and is critical for cellular adaptation and survival under changing conditions. Therefore, an organism's health depends not only on mitochondrial energy production, but on the ability to sense metabolic status and the ability of mitochondria to signal appropriately to use that energy. 1,2 The mechanisms through which cellular energy-related signaling can occur likely depend on the electrochemical gradient across the mitochondrial inner membrane (IM). Mitochondrial respiration results in a proton gradient across the IM known as the protonmotive force (PMF). Ultimately, respiratory complexes of the electron transport chain (ETC) convert chemical energy into electrical potential energy by pumping protons across the IM, creating this gradient. The PMF can then be consumed at ATP synthase,

BACKGROUND: Heparin is the standard anticoagulant for cardiopulmonary bypass (CPB); however, there are problems with its use that make the development of suitable alternatives desirable. Currently, no ideal alternative exists. We have previously reported that the direct thrombin inhibitor dabigatran can prevent coagulation in simulated CPB at high concentrations. These high concentrations may cause difficulties in achieving the reversal of dabigatran with idarucizumab, given the markedly different pharmacokinetics of the 2 drugs. Herein, we test the hypothesis that the addition of the anti-Xa drug rivaroxaban would provide suitable anticoagulation at a lower concentration of dabigatran given likely synergy between the 2 classes of drugs. The primary goal of the study was to investigate whether the addition of rivaroxaban reduces the concentration of dabigatran necessary to allow 2 hours of simulated CPB. METHODS:The study was performed in sequential steps. Blood collected from consenting healthy donors was used throughout. First, we added graded concentrations of dabigatran and rivaroxaban alone and in combination and assessed inhibition of anticoagulation using thromboelastometry. Using results from this step, combinations of dabigatran and rivaroxaban were tested in both Chandler loop and simulated CPB circuits. Dabigatran and rivaroxaban were added before recalcification, and the circuits were run for 120 minutes. In both models of CPB, 120 minutes of circulation without visible thrombus was considered successful. In the Chandler loop system, idarucizumab was added to reverse anticoagulant effects. In the CPB circuits, the arterial line filters were examined using scanning electron microscope (SEM) to qualitatively assess for fibrin deposition. RESULTS: In vitro analysis of blood samples treated with dabigatran and rivaroxaban showed that dabigatran and rivaroxaban individually prolonged clotting time (CT) in a dose-dependent manner. However, when combined, the drugs behaved synergistically. In the Chandler loop system, dabigatran 2400 and 4800 ng/mL plus rivaroxaban (150 ng/mL) effectively prevented clot formation and reduced the dynamics of clot propagation for 120 minutes. Idarucizumab (250-1000 µg/mL) effectively reversed anticoagulation. In the CPB circuits, dabigatran (2500 ng/mL) and rivaroxaban (200 ng/mL) were successful in allowing 120 minutes of simulated CPB and prevented fibrin deposition. Biomarkers of coagulation activation did not increase during simulated CPB. Heparin controls performed similarly to dabigatran and rivaroxaban. CONCLUSIONS: The dual administration of oral anticoagulant drugs (dabigatran and Rivaroxaban) with different pharmacologic mechanisms of action produced synergistic inhibition of coagulation in vitro and successfully prevented clotting during simulated CPB. (Anesth Analg 2022;135:52-9) KEY POINTS• Question: Do lower concentrations of dabigatran administered with rivaroxaban prevent clotting during simulated cardiopulmonary bypass (CPB)? • Findings: Dabigatran and rivarox...

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

28Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial 29 inner membrane called the protonmotive force (PMF) to drive diverse functions and make ATP. 30 Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, 31 reversible method to increase the PMF. To address this issue, we used an optogenetic 32 approach and engineered a mitochondria-targeted light-activated proton pumping protein we 33 called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the 34 PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial 35 toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient 36 mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic 37 preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both 38 necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that 39 optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling 40 outcomes. 41 42 76organism Leptosphaeria maculans (Chow et al., 2010, Waschuk, Bezerra et al., 2005 to 77 3 mitochondria and selectively increase the PMF. We call this optogenetic tool mitochondria-ON 78 (mtON) due to its ability to mimic the proton pumping activity of the ETC in response to light, 79 independent of oxygen or substrate availability. 80 We validated mtON using the well-characterized genetic model organism, C. elegans 81 (Butler, Ventura et al., 2010, Dingley et al., 2010, Tsang & Lemire, 2003. Using hypoxia and 82 reoxygenation, we tested the hypothesis that hypoxia adaptation through preconditioning 83 requires a decreased PMF. Our data demonstrate that transient loss of PMF during 84 preconditioning is necessary and sufficient for resistance to hypoxia. By probing the 85 evolutionarily conserved hypoxia adaptation response (Pena, Sherman et al., 2016, Wang, Lim 86 et al., 2019, Wojtovich, Nadtochiy et al., 2012b, Wojtovich, Nadtochiy et al., 2013, we show that 87 tools like mtON allow precise determination of cause and effect in physiologic models. 88 89 RESULTS & DISCUSSION 90 Light-activated proton pump mitochondria-ON (mtON) is expressed in mitochondria. 91 Using a ubiquitously-expressed gene promoter (Peft-3), we directed expression of a 92 light-activated proton pump to the mitochondrial IM in C. elegans. Mitochondrial localization was 93 achieved by fusion of the proton pump to an N-terminal mitochondrial targeting sequence of the 94 IMMT1 protein (Fischer, Igoudjil et al., 2011, John, Shang et al., 2005 in an orientation that 95 allows proton pumping from the mitochondrial matrix towards the intermembrane space to 96 increase the PMF in response to light (Fig. 1A). Using a C-terminal GFP fusion for subcellular 97 visualization, and MitoTracker TM CMXRos, we observed overlap of green and red fluorescence 98 in C. elegans tissues, indicating the intended mitochondrial...

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