ATF5 mitoUPR pre-print

Our paper on the role of ATF5 in the mammalian mitochondrial unfolded protein response (UPRmt) is now on BioRxiv as a pre-print. It’s also been submitted to a regular journal, so hopefully will be in press some time this millenium.

For the uninitiated, UPRmt is a mitochondrion specific unfolded protein response, much of which was figured out in worms (C. elegans). The key mediator is the transcription factor ATFS1, which has both mito’ and nuclear targeting domains. It is normally made and imported into mito’s and destroyed by proteases. When mito’ proteostasis is upset (e.g., a mis-match in stoichiometry between the mtDNA and nuclear DNA encoded subunits of the electron transport chain, which causes the leftover bits to mis-fold or aggregate), the import is blocked and ATFS1 goes to the nucleus. There, it upregulates a bunch of things to restore mito’ homestasis including chaperones, antioxidants, and glycolysis to take care of energy needs while the mito’s are undergoing repair.

All those things (chaperones, antioxidants, glycolysis) sound like they might be useful to have around in a situation such as cardiac ischemia (i.e., what we study), so we reasoned that activating mito-UPR might be cardioprotective. To induce it in mice, we used oligomycin (yes, that ATP synthase inhibitor) or doxycycline (a tetracycline antibiotic that disrupts mito’ ribosomes – which is a good reason not to use it in experiments). Sure enough, after 6 hours (as expected for a gene program) the hearts were protected against ischemia-reperfusion injury.

Within the mito’ UPR field there’s a debate about what the mammalian ortholog of the worm ATFS1 protein is. Some folks have said the mito UPR is perhaps just a branch of the integrated stress response, mediated mainly by ATF4. This has been accompanied by a general perception in the field that maybe mito UPR is just a worm thing and might not really exist in mammals at all?  That changed a couple of years ago when our collaborator Cole Haynes showed that the mammalian trasncription factor ATF5 can rescue the ATFS1 knockout worm.

So, we tested the UPRmt inducers for cardioprotection in an Atf5-/- mouse, and the protection was lost. To the best of our knowledge, this is the first in-vivo demonstration that ATF5 is a component of the mito’ UPR in mammals (previously it had been shown in cell culture).  We also did some RNA-Seq, trying to find out the pathways induced, and while we did see the classical UPRmt target genes such as hsp60 were up by qPCR, there wasn’t really much going on in the seq’ data – certainly no gene signatures or big pathways. In some ways this is a good thing – it would be kinda boring if we just saw an ISR or Nrf2/Keap or NFKB or any of those other “classical” stress signatures. Instead, it’s likely we’ll have to drill down at the individual target level to really identify the downstream molecular mechanism by which ATF5 is inducing protection.

The other interesting aside to this paper, is there’s quite a sizeable literature on the use of tetracycline antibiotics for cardioprotection, including some human clinical trials. To date, the party line has been that doxycycline confers protection by inhibiting matrix metalloproteases (they’re induced during and after heart attack). But, our data showing a requirement of ATF5 for dox-induced protection suggests an alternative mechanism.

Finally, a note on the practicalities of this operation.  The paper’s lead author, Yves Wang, undertook a Herculean effort just to get the study done, because the Atf5-/- mice are a total nightmare to deal with!  Due to an olfactory neural defect, the homozygotes exhibit ~80% neonatal mortality – the pups can’t find the teat and so can’t get milk. As such, the vivarium bill for maintaining the Atf5-/- colony is currently running about $2500 a month, even with our exceptionally low per-diem rates. Hopefully once this is published we can down-size a bit!


Papers, Graduations & More

Lots of recent happenings in the lab to report…

(1) Congratulations to Owen Smith, who successfully defended his PhD thesis in March!  Owen’s final paper from his thesis work is currently in revision, but you can see the pre-print at BioRxiv here.  Owen is going on to do a post-doc in the lab of Roman Eliseev in the Center for Musculoskeletal Research.

(2) On April 11th we have another graduation – Jimmy Zhang is finishing up the PhD part of his MD/PhD and returning to medical school.  Jimmy’s last paper on his thesis work is also in revision, but no pre-print yet as per journal policy (boo!)  The short version – not everything in this paper appears to be 100% correct.

(3) We have a new post-doc’ in the lab. Chaitanya Kulkarni (Chaitu) came from Bob Kern‘s lab in the Medicinal Chemistry program at the University of Iowa, with a strong background in developing mito-targeted drugs.

(4) Our paper on the metabolic effects of NMN is also in revision, but the BioRxiv pre-print is here.

(5) Some work on metabolomics of the developing embryonic heart, performed with our collaborators Steve Ebert at UCF and George Porter here in Rochester was published in J Biol Chem.

With another paper about to be submitted, all being well we should be looking at hlf a dozen primary science manuscripts in press by late summer, plus a couple of reviews. Publish or perish…

Latest Paper – Mito’ KNa1.2 Channel

Our paper on a mitochondrial KNa1.2 channel is now out as a pre-print at BioRxiv. It describes about 4 years of work by Owen Smith, a (hopefully soon to be graduated) student in the lab.

This new report builds off our paper last year showing that KNa1.2** is required for cardioprotection by volatile anesthetic preconditioning (APC). In that paper, we kind-of showed that there’s a mitochondrial KNa1.2 channel, but the gold standard is patch-clamp of isolated mito’ inner membranes (mitoplasts) and, to put it mildly, this is not a simple technique! With the help of Liz Jonas at Yale and Casey Kinally at NYU (now retired), Owen finally cracked it and we now have solid evidence the channel exists in mitochondria.

The next question was “OK, if this channel exists, it didn’t evolve over millions of years waiting for volatile anesthetics to be invented in the 20th century, so what’s its endogenous physiologic role?”  What we found is that the hearts of channel knockouts have a rather odd metabolic phenotype – they can’t exhibit maximal respiratory or work capacity, BUT this is only true when they’re burning fat as a fuel. They’re perfectly OK on glucose, and they’re fine at baseline, they just can’t go full-speed when burning fat.

This finding has some interesting implications…. First, it implies a mechanistic link between a mitochondrial potassium channel and the regulation of cardiac metabolism. To the best of our knowledge this is the first such reported link.  Second, we found that activating mito’ KNa1.2 uncouples mitochondria, and it was reported in 2014 that Niclosamide, a KNa activator, is beneficial in a high fat diet model of diabetes. Ergo, this mito’ channel could be an important (and so far overlooked) drug target for regulating metabolism, with potential importance for obesity, diabetes, metabolic syndrome etc.

Anyway, while the paper is being reviewed at a “regular” journal (fingers crossed), the pre-print hopefully gets the story out there for critique by the field at large. If you have something to say  – have at it in the BioRxiv comments, or Tweet/E-mail me. Our previous pre-prints have benefitted enormously from incorporation of comments on BioRxiv at the journal revision stage.

**For those confused by the naming, KNa1.2 is the new name for “Slo2.1”, which is also known as “Slick”, and is encoded by the Kcnt2 gene. We used to call it by the Slo nomenclature, since Slo2.1 is part of a larger family of channels related to the Drosophila slow-poke allele, which is Slo1. Slo1 is a KCa channel, whereas Slo2.1 and its sibling Slo2.2 (aka KNa1.1, Slack) are KNa channels.

New grant, new toys, conferences, and we’re hiring!

Some exciting recent developments in the lab…

GRANTS!  Our long-running NIH R01 project (R01-HL-071158) was competitively renewed for its 4th cycle (through June 2021).  The current iteration of the project aims to understand the role of acidic pH in remodeling of cardiac metabolism that occurs in ischemia. Here’s the project description on NIH RePorter.

RESULTS!  Our paper on the role (or not!) of sirtuin-dependent deacetylation in regulating 2-hydroxyglutarate generation was published in Biochem J.  We have another paper in the pipeline on the regulation of metabolism by sirtuins, to be submitted this fall.

TOYS!  The financial stars aligned, and we were fortunate to get the opportunity to acquire a new (well, refurbished) mass-spec’ for the lab.

It‘s a Thermo TSQ Quantum Access Max, which is their work-horse triple-quad. It’s plumbed in next to our HPLC, and will enable us to perform metabolomic analyses in-house (although our colleague Josh Munger remains an important collaborator, having gotten us into this field to begin with!)   Our new lab Tech’ Mary is currently developing a custom SRM library for targeted metabolomics (aka making us bankrupt by purchasing the entire Sigma catalog!)


PEOPLE! Our GEBS summer student Irma Ruiz finished up and is now back at Tulane.

Irma worked on optimizing and running blue-native and clear-native gels, for the study of mitochondrial respiratory super-complexes, making some interesting findings in the various knockout mouse lines we maintain.



MEETINGS!  Lab members will be presenting at a few upcoming conferences…

JOIN US!  Coupled to the grant competing renewal, we’re looking to hire a new post-doctoral fellow to work on cardiac metabolomics. Here is a copy of the job ad’, which will be posted at the various meetings listed above. If you’re interested, feel free to talk with any of us during these meetings, or call or email for more details.

New Tech, Student & Papers in the pipeline

We have a new lab’ tech – Mary Day – who joined us from SUNY Oswego via the UR clinical labs). Mary will be taking over the management of our mouse colonies, among other things.

We also have a student in the GEBS summer scholar’s program – Irma Ruiz-Concepcion, from Tulane University in New Orleans, who will be looking at respiratory super-complexes in mouse hearts.

Our latest paper on the the role (or not!) of acetylation as a regulator of 2-hydroxyglutarate generation, is now up as a pre-print on BioRxiv.  Also our collaborator Paul Trippier’s paper on novel complex II inhibitors was accepted. We also have a number of other papers in the pipeline this summer, from colaborations with Michael O’Reilly, Xin Li, Gail Johnson, Vadim Ten, and Daniel Acuna. Plus a chapter on mitochondria and nitric oxide in the upcoming 3rd edition of Ignarro’s “NO Bible“, co-authored with Sruti Shiva and Laura Castro.  The Biochem. J. review on mito’ K+ channels should be out any day now.