Research in the lab’ is supported by the following:
RO1 HL-071158 “SIRT1, Acidosis & Metabolism in Cardioprotection”. 7/01/2017-06/30/2021 (originally funded 8/2003, currently on 4rd cycle).
RO1 GM-087483 “Mitochondrial K+ Channels & Cardioprotection”. 01/01/2014-12/31/2017. (Joint PI grant with Keith Nehrke, originally funded 1/2010, currently on no cost extension).
RO1 HL-127891 “The role of mitochondrial UPR in ischemic protection” 04/01/2015-03/31/2019 (Joint PI grant with Keith Nehrke & Cole Haynes at UMass Med Ctr.)
Our broad research interest is cardioprotection against ischemia-reperfusion (IR) injury. We are particularly interested in mechanisms of protection in ischemic preconditioning (IPC), and anesthetic preconditioning (APC) and the role of mitochondria and metabolism in these processes. A variety of model systems are used, including: isolated heart mitochondria, Langendorff perfused mouse hearts, isolated adult mouse cardiomyocytes, in-vivo mouse coronary artery occlusion, and H9c2 cardiomyocytes in cell culture. We also use many biochemical techniques to investigate mitochondrial function including: mitochondrial respiration & membrane potential assays, electrode and fluorescence based measurements of reactive oxygen species (ROS) and nitric oxide, analysis of protein post-translational modifications (phosphorylation, acetylation, S-nitrosation, nitroalkylation) by 2D gels, western blotting, mass spectrometry (via the proteomics core), spectrophotometry, fluorescence spectroscopy, Seahorse XF24 and XF96 extracellular flux analysis, chemical synthesis and development of novel small molecule therapeutics, and metabolomics (with Josh Munger‘s lab). We maintain several lines of engineered mice for these studies (email for details).
Current Active Projects (no particular order of priority)
Sirtuins, Metabolism & Cardioprotection
Sirtuins (SIRTs) are a family of NAD+ dependent lysine deacylases. We showed, using SIRT1 knockout and overexpressing mice as well as pharmacologic agents, that cytosolic SIRT1 is both necessary and sufficient for acute IPC. More recently we reported that the metabolic remodeling that occurs in IPC is also dependent on SIRT1. We also have an interest in SIRT3 and its role in the loss of cardioprotection in aging.
During these studies, a metabolite of interest that emerged was 2-hydroxyglutarate (2-HG). Originally viewed as an oncometabolite, we were among the first labs to associate 2-HG with hypoxia. We went on to show that acid pH is the driver of 2-HG generation in hypoxia (a finding subsequently confirmed by other labs). Current studies are aimed at further understanding the role of acidic pH in the remodeling of metabolism during ischemia, and at identifying the downstream signaling targets if 2-HG (i.e. α-KG dependent dioxygenases).
Mitochondrial Potassium Channels, Cardioprotection & Metabolism
Volatile anesthetics (e.g., isoflurane) protect the heart against IR injury in a process termed “anesthetic preconditioning” (APC). A mitochondrial K+ channel is thought to mediate APC, and until recently the field had focused on the channel encoded by the gene Slo1. We showed that Slo1-/- knockout mice can still be preconditioned, and their mitochondria contain a K+ channel acitivity. Subsequently, we reported that a different channel (encoded by the gene Slo2.1) is required for APC.
Since Slo2 channels are evolutionarily conserved (from C. elegans to H. sapiens) they did not evolve over millions of years for the purpose of responding to volatile anesthetics! Ongoing work is aimed at characterizing the normal physiologic function of SLO2.1. We have idenfitied a metabolic phenotype in the Slo2.1-/- mouse, suggesting these channels have a role in regulating mitochondrial metabolism. We are also interested in characterizing the electrophysiologic properties of mito’ K+ channels, using patch-clamp on mitoplasts (isolated inner membranes), in collaboration with Bob Dirksen in Rochester and Liz Jonas at Yale.
The Mitochondrial Unfolded Protein Response (UPRmt) & Cardioprotection
A bi-functional transcription factor, ATFS-1, contains both mitochondrial and nuclear targeting sequences. Under normal conditions, ATFS-1 is imported into mitochondria and destroyed by proteases. Under conditions of mitochondrial stress or protein misfolding (mito-nuclear protein imbalance), ATFS-1 import is blocked, sending it to the nucleus where it upregulates synthesis of mitochondrial chaperones. We are interested in the possibility that activation of the UPRmt may protect the heart from IR injury. Recently, the mammalian homolog of ATFS-1 has been identified as ATF5, and we are investigating a cardioprotective role for UPRmt using Atf5-/- mice. In addition, we are interested in the manner in which the transcriptional readout of the UPRmt can be modulated by metabolic epigenetic effectors.
Succinate in Ischemia
We have a long-standing interest in the use of inhibitors of the mitochondrial respiratory chain to protect organs against IR injury. We were part of a multi-national collaboration with Mike Murphy’s group (Cambridge UK) on a paper that identified succinate accumulation during ischemia as a major driver of ROS generation at reperfusion. More recently, we have been probing the precise biochemical mechanisms of ischemic succinate accumulation, using 13C isotope labeled metabolite tracer experiments. We also have an interest in the therapeutic use of mitochondrial complex II inhibitors in IR injury, including a collaboration with Paul Trippier at Texas Tech’ School of Pharmacy, who has developed a series of novel compounds.