Research in the lab’ is supported by the following:
RO1 HL-071158 “SIRT1 & Nitro-Lipids in Cardioprotection”. 7/01/2013-06/30/2017 (originally funded 8/2003, currently on 3rd 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 2nd cycle).
RO1 HL-127891 “The role of mitochondrial UPR in ischemic protection” 04/01/2015-03/31/2019 (Joint PI grant with Keith Nehrke)
Our broad research interest is cardiac ischemia-reperfusion (IR) injury and cardioprotection. We are particularly interested in the mechanisms of protection elicited by ischemic preconditioning (IPC), and anesthetic preconditioning (APC) and the role of mitochondria and nitric oxide in cardioprotection.
A variety of model systems are used, including: isolated heart and liver mitochondria, Langendorff perfused mouse and rat hearts, isolated adult rat 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 functional analyses (respiration, membrane potential), 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), high resolution spectrophotometry, fiber optic based fluorescence spectroscopy, Seahorse XF24 and XF96 extracellular flux analysis, chemical synthesis and development of novel therapeutic molecules, and metabolomics (with Josh Munger‘s lab). We maintain several lines of engineered mice for these studies (email for details).
Active Projects (in no particular order of priority)
Mild mitochondrial uncoupling, nitroalkenes & cardioprotection.
Long ago we found that mitochondria from hearts exposed to IPC exhibited a small but significant increase in their membrane proton leak, mediated by carrier molecules in the inner membrane (adenine nucleotide translocase, ANT, and uncoupling proteins, UCPs). Small amounts of mitochondrial uncoupling can be beneficial, acting like a “safety valve” to prevent excess generation of ROS, and consistent with this both overexpression of UCPs and miniscule doses of chemical uncouplers can elicit cardioprotection. Furthermore, electrophilic lipids can activate mild uncoupling by acting on ANT and UCPs.
More recently, nitro-lipids (fatty acid nitroalkenes, e.g. nitrolinoleate) have been discovered as a novel class of endogenous anti-inflammatory lipid signaling molecules. We discovered that nitrolinoleate induces mitochondrial uncoupling, and is potently cardioprotective. In collaboration with Bruce Freeman at Pittsburgh we showed that nitrolinoleate is formed endogenously in mitochondria during IPC. Furthermore, nitrolipids are electrophilic, and can modify cysteine residues in proteins, and werecently demonstrated that nitro-linoleate covalently modifiesCys-57 on ANT and this is required for its cardioprotective activity. We are currently exploring the downstream mechanisms by which nitro-lipids and mild uncoupling can afford protection against ischemic injury, including via activation of mitophagy. We have also developed mito-targeted nitro-lipids to enhance the specificity of these molecules.
Cardioprotection by Sirtuins
The sirtuins (SIRTs) are a family of lysine deacetylases, requiring NAD+ as a substrate. Consensus holds that lysine acetylation is an important post-translational modification, and that SIRTs may serve a “metabolic sentinel” role in the cell, to regulate metabolism. We showed, using SIRT1 knockout and overexpressing mice (as well as pharmacologic agents), that cytosolic SIRT1 is both necessary and sufficient for acute ischemic preconditioning, and that changes in the the acetylation status of several important metabolic proteins may underlie the protective signaling effects of SIRT1. Notably, SIRT1 is proposed as a mediator of the lifespan-extending effects of caloric restriction, and as a target for red wine polyphenol resveratrol, although both these aspects of SIRT function are incredibly controversial. We are currently investigating theupstream regulation and downstream targets of SIRT1 in IPC. We also have an interest in SIRT3 and its role in the loss of cardioprotection in aging. Recently, using metabolomics analysis, we reported on the role that SIRT1 plays in regulating short-term metabolic changes that occur in the IPC heart.
Mitochondrial KCa channels in anesthetic preconditioning
Volatile anesthetics (isoflurane, desflurane, sevoflurane etc.) can protect the heart against IR injury in a process termed anesthetic preconditioning (APC). A large conductance (BK) Ca2+ sensitive K+ channel in the mitochondrion is proposed to mediate APC, in the same manner that a mKATP channel is implicated in IPC (see below). To date, the field has focused on the SLO-1 isoform of BK, but we recently showed that SLO-1 knockout mice can still be preconditioned by isoflurane, and their mitochondria contain a BK channel acitivity. Furthermore, knockout of the SLO-2 channel isoform abolishes APC in C. elegans. We are have subsequently pursued SLO-2 variants as candidate mitochondrial BK channels in mammals, and recently reported that Slo2.1 is required for APC. Furthermore, we are interested in the mechanisms of cardioprotection by BK channel activators such as NS1619, and have recently shown that these molecules in-fact work via BK channels in intrinsic cardiac neurons. Spearheaded by graduate student Owen Smith, we have been working with Bob Dirksen in Rochester and Liz Jonas at Yale, to perform electrophysiology studies (patch clamp) on mitoplasts (isolated inner membranes) from mitochondria of various K+ channel knockout mice, to conclusively identify mitochondrial K+ channels. In addition to APC, we are working on the baseline function of these channels (i.e. what do they do in mitochondria while waiting around to participate in cardioprotection?)
Mitochondrial ATP sensitive K+ channels
A consensus is held that a mitochondrial ATP sensitive K+ channel (mKATP) is central to the mechanism of cardioprotection by IPC. However, the molecular identity of this channel is unknown. We are currently investigating this channel and its role in cardioprotection from a number of angles: First, we are interested in cross-talk between the channel and complex II of the mitochondrial respiratory chain. We found that the complex II inhibitors atpenin A5 and malonate can open mKATP, and are using these and other molecules as tools to probe mKATP function. Second, we discovered that the genetic model organism C. elegans has a mKATP channel. Furthermore, we recently showed that the mKATP required for hypoxic preconditioning in the worm does not originate from the KIR gene family typically associated with KATP channels, suggesting mKATP may originate from a different channel family. Third, we are interested in regulation of mKATP channel, particularly by redox and how this relates to channel function in IPC. Also, we have developed a novel method to assay mKATP channel activity based on the uptake of thallium (Tl+) into mitochondria as a surrogate for K+ flux.
High-Throughput Screening (HTS) for cardioprotection
There are currently no FDA approved drugs for the treatment of AMI, acute myocardial infarctio, and there have been some high profile failures in late phase clinical trials for mechanism-based therapeutics (i.e. drugs designed to hit a specific cellular target). Phenotypic HTS is a “mechanism agnostic” drug discovery approach, in which a phenotypically relevant model and end point are used to screen a diverse chemical library, without regard to mechanism or target.
By adapting the interior mechanical systems of the Seahorse XF platform for gas flow, we developed a 24 well cell-based model of IR injury, in which pO2, pH, and metabolic function are measured in every well. We used this system to screen a 2000 compound library and identified a number of protective molecules. Hits from the screen were validated using the Langendorff perfused heart model of IR injury. We are currently both pursuing hits to determine mechanism, and developing new screens to probe the relationship between metabolism and cardioprotection. This assay also forms an important platform for testing cardioprotection in many other projects in the lab. In 2013 we obtained a Seahorse XF96, as part of a consortium of investigators and with support from the URMC SAC equipment fund, and we are now adapting this instrument for gas flow studies too.
Role of S-nitrosation and complex I in cardioprotection.
We established that Complex I of the respiratory chain is a bona fide target for modification by S-nitrosation. This modification reversibly inhibits mitochondrial respiration, and is also known to occur during IPC, suggesting it may play a role in cardioprotection. To exploit this, we have developed a series of mitochondrial S-nitrosating agents, of which S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) has shown promise in models of cardiac IR injury. Specifically, SNO-MPG protects perfused hearts against IR injury at 10 µM, and protects the mouse heart in-vivo at a dose of 1mg/kg. Notably, complex I was essential for protection, since protection was lost in a complex I knockout animal. Importantly, SNO-MPG is also protective when delivered at reperfusion, a more clinically relevant scenario. Current interests in this project include testing of additional compounds (including a more potent molecule MitoSNO1 from our collaborator Mike Murphy in Cambridge), and determining the possible role of S-nitrosation in other paradigms of cardioprotection, such as that afforded by nitrite.
“Back Burner” projects and other areas of interest:
Gradual wake-up” from IR injury
Our work on complex I fits into a larger project regarding the role of reversible mitochondrial respiratory inhibition in cardioprotection. At reperfusion, cardiac mitochondria experience an onslaught of ROS generation and Ca2+ overload, which is bought about by a rapid re-establishment of respiration (within 5 s. of restoration of blood flow). It is apparent that a multitude of reversible mitochondrial inhibitors are cardioprotective, and this protection appears to proceed by facilitating a more gradual re-starting of mitochondrial metabolism, this avoiding a surge of pathologic ROS and Ca2+. We are interested in other mechanisms by which reversible inhibitors can be cardioprotective, and were fortunate to recently collaborate with Mike Murphy’s group (Cambridge UK) on a project that identified succinate accumulation as a major driver of post-ischemic ROS generation.
Mitochondria, Redox and Cancer
We maintain an active collaboration with Steven Bernstein in the Wilmot Cancer Center at URMC, regarding the mechanisms of action of various electrophilic anti-lymphoma agents. We recently showed that modulation of cell surface thiol status on cancer cells may be an important but overlooked mechanism of cell death induction by electrophiles. We also have a long-standing interest in protein folding in the mitochondria, and showed that the mitochondrial protease Lon is both sensitive to redox modifiers, and is an important vorulence factor in cancer cells. We also have an active collaboration with several investigators to use the Seahorse XF platform for measuring metabolism of cancer cells, which led to the discovery of a novel role for p90RSK in the “Crabtree effect” (a congener of the Warburg effect).
Mitochondrial ROS signaling in hypoxia
The lab has a long interest in mechanisms and regulation of mitochondrial ROS generation, and we are particularly interested in the effect of hypoxia. A paradox exists, wherein cells exposed to hypoxia appear to increase their mitochondrial ROS generation. How can mitochondria make more ROS when the substrate for making ROS (oxygen) is less abundant? Proponents of more ROS in hypoxia suggest this phenomenon is an autonomous property of the mitochondrial respiratory chain. Using the technique of open-flow respirometry, which allows us to maintain isolated mitochondria at very tightly defined O2 tensions, we definitively showed that ROS generation by isolated mitochondria follows a hyperbolic relationship, i.e. it is lower in hypoxia. Thus, we conclude that the mitochondrial respiratory chain does not automatically produce more ROS when oxygen is low – instead we think that another signaling component in cells is responsible for up-regulating mitochondrial ROS in hypoxia. More recent work has shown that the apparent kM for oxygen of the many ROS generating sites within mitochondria varies considerably. Thus, the importance of these different ROS generating sites under various pathological conditions may have been mis-estimated due to the use of non-physiological oxygen tensions, for example in cell culture experiments.
Mitochondrial NOS (mtNOS)
A raging debate surrounds the existence of a specific and unique isoforms of nitric oxide synthase (NOS) within mitochondria. Following my publication of a negative review on this topic in 2004, a number of other prominent researchers also published negative data, and expressed concerns about the validity of the data and flawed methodology. More recently it was proposed that a mammalian ortholog of a plant NOS might be mtNOS, but the foundation work establishing the identity of the plant protein as a NOS has since been retracted (the protein, hatNOA1 is actually a GTPase). Despite much evidence to the contrary, the notion that mitochondria contain a unique isoform of NOS still persists in the literature today.