December 2022 Discover CircRes

This month on Episode 43 of Discover CircRes, guest host Nicole Purcell highlights two original research articles featured in the December 2 issue of Circulation Research. This episode also features an interview with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.   Article highlights:   Akerberg, et al. RBPMS2 Regulates RNA Splicing in Cardiomyocytes   Lv, et al. Cardiac Protection by MG53-S255A Mutant   Nicole Purcell:             Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I am your host, Dr Nicole Purcell, from the Huntington Medical Research Institutes in Pasadena, California, and today I will be highlighting two articles from our December 2 issue of Circulation Research. I'll also have a chat with Drs Aaron Phillips and Kevin O'Gallagher about their study, The Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans.   Nicole Purcell:             But before I get to the interview, here are a few article highlights. The first article we're going to highlight is RBPMS2 Is a Myocardial Enriched Splicing Regulator Required for Cardiac Function. This comes from Boston Children's Hospital with first author Dr Alexander Akerberg, and corresponding author Dr Jeffrey Burns. RNA splicing, along with transcription control and post-translational modifications, is a mechanism for fine tuning the expression of a gene for a particular purpose in a particular tissue. Factors that control splicing are thus often enriched in certain cell types. The factor, RBPMS2, for example, is enriched in the myocytes of amphibians, fish, birds and mammals.  This conserve tissue specificity suggesting essential role of RBPMS2 in heart function.   Akerberg and colleagues now confirm this is indeed the case. They generated zebra fish embryos and human cardiomyocytes lacking RBPMS2, and found the fish suffered early cardiac dysfunction by 48 hours post fertilization. The animal's hearts had reduced ejection fractions, compared with the hearts of controlled fish. At the cellular level, the RBPMS2 lacking fish cardiomyocytes displayed malformed sarcomere fibers and disrupted calcium handling, both of which were also seen in the RBPMS2 deficient human cardiomyocytes. Furthermore, RNA sequencing experiments revealed a conserve set of 29 genes in the RBPMS2-lacking fish and human cells that were incorrectly spliced. In revealing the essential cardiac role of RBPMS2 and its RNA targets, the work provides new molecular details for understanding vertebrate heart function and disease, say the team.   Nicole Purcell:             Our second article being highlighted is Blocking MG53 Serine 255 Phosphorylation Protects Diabetic Heart from Ischemic Injury. This comes from Peking University with first authors, Fengxiang L, Yingfan Wang and Dan Shan, as well as corresponding author Dr Rui-Ping Xiao. Midsegment 53, or MG53, is a recently discovered muscle-specific protein that is an essential component of the cell membrane repair machinery with cardioprotective effects. MG53 thus has therapeutic potential, but for patients whose heart disease is linked to type 2 diabetes, there's a problem. MG53 also tags certain cellular proteins for destruction, including the insulin receptor and the insulin signaling factor, IRS1. Loss of these factors could worsen insulin resistance. lev and colleagues therefore investigate whether MG53 could be tweaked to provide protection without the diabetes downside.   Nicole Purcell:             They discovered the phosphorylation of MG53 at serine 255 is required for its role in protein destruction, and that a mutant version of MG53, incapable of this phosphorylation, MG53 serine to 255 alanine mutant, could still promote cardiomyocyte survival, and protect the cells from membrane damaging insults. Importantly, when a diabetic mouse model was injected with MG53 serine 255 to alanine mutant, the protein better protected the animals against myocardial infarction than injection with the wild type MG53, recipients of which had poor insulin sensitivity. Based on these findings, the authors suggest MG53 serine 255 alanine mutant could be developed into a heart protective drug, for use in diabetic and non-diabetic patients alike.   Nicole Purcell:             Today, Dr Aaron Phillips and Dr Kevin O'Gallagher from University of Calgary are with me to discuss their study, the Effect of a Neuronal Nitric Oxide Synthase Inhibitor on Neurovascular Regulation in Humans in our December 2 issue of Circulation Research. Thank you for joining me today.   Kevin O'Gallagher:    Hello, my name's Dr Kevin O'Gallagher. I'm a British Heart Foundation clinician scientist and interventional cardiologist at Kings College London and Kings College Hospital NHS Foundation Trust.   Aaron Phillips:            Hello, my name's Dr Aaron Phillips. I'm an associate professor in physiology, pharmacology, cardiac sciences, biomedical engineering and clinical neurosciences at the University of Calgary in the Hotchkiss Brain Institute and Libin Cardiovascular Institute. I am also the director of the Restore Network, which is a large platform at the University of Calgary spanning all these groups, developing new tools and techniques for translational research into neurological conditions.   Nicole Purcell:            There are a lot of authors involved in this study. While all could not join us, I appreciate you taking the time to discuss your findings today. Your paper deals with looking at neurovascular control in humans. Two primary regulatory pathways are neurovascular coupling, or NVC, and dynamic cerebral autoregulation. Dr Phillips, can you explain what NVC to our audience, and what does dysregulation lead to?   Aaron Phillips:            Yeah, thanks Nicole and I'm happy to be here. Thank you for the invitation. NVC, or neurovascular coupling, we've been studying it for about 15 years. At its fundamental level, it's kind of this elegant interplay between neurons, which unfortunately have very limited capacity for substrate storage. The brain has very limited substrate storage capacity, and so neurons need to very rapidly match their metabolic activity to the blood flow that's being delivered to them, and that needs to happen locally, for areas of the brain that have greater metabolic needs as opposed to other areas.   What happens, in terms of dysregulation or conditions that are associated with dysregulation, it's an interesting story because we still really need to understand the mechanisms fully, in order to suss out what clinical conditions should have dysfunction of this unit. We know that certain conditions, such as vascular cognitive impairment, even spinal cord injury, we've done some work in stroke patients, it seems to be dysfunctional in all of these conditions, but understanding exactly why it's dysfunctional, we're still establishing that.   Nicole Purcell:             Great. You were talking about how it's the connection or interplay between blood flow, so we're talking about altered blood pressure seems to play a key role in neurovascular coupling. So, for those listeners not familiar with this field, can you explain how nitric oxide synthase and its isoforms, how this relates to NVC?   Aaron Phillips:            Well, nitric oxide synthase is an enzyme that produces nitric oxide that's expressed primarily in neurons. Nitric oxide is a powerful vasodilator. It actually works on quite a rapid time course. So, we surmised, we suspected, and there were some preclinical work before our human study, that neuronal sources of nitric oxide, being that nitric oxide is a potent vasodilator, we thought that would be likely to be mediating a large part of the neurovascular coupling response.   Nicole Purcell:             Great. So, Dr O'Gallagher, based on that, what was your main objective or hypothesis of this study, and how is your study novel from those that have already just suggested, looked at NOS regulation for cerebral blood flow?   Kevin O'Gallagher:    Thanks very much for the invite to talk. I mean, we hypothesized that nNOS would have a role in regulating neurovascular coupling. I think the novelty of our study is that although people have been interested in NOS and its regulation of cerebral vascular and cardiovascular blood flow, it's only relatively recently that there has become an agent available that will specifically inhibit nNOS, and therefore give us an idea of what it is doing, rather than previous inhibitors which just inhibit all of the three NOS isoforms. It was really that the development of the agent was what allowed us to do this study. I think it was really through that, that makes this an interesting finding that nNOS does play a role in neurovascular coupling, and really pushes the field forward ever so slightly.   Nicole Purcell:             Great. So, as you pointed out, this is a specific nNOS inhibitor, which is known as SMTC. It's a synthetic L-Arginine analog, right? That's really what sets your study apart. Can you tell us a little bit the audience, whether that be you, Dr Phillips or Dr O'Gallagher, about what your study was and what did you find, and how did an ambition of using this SMTC to inhibit nNOS affect systemic hemodynamic changes and NVC?   Aaron Phillips:            Yeah, I think both of us can probably speak to this interchangeably and add in different elements of the experiment. This is kind of a summary of the study, I guess. In advance of this, adding on what Kevin had just said in terms of the novelty of the study and the importance, we had done a lot of work previous to this paper where we were one of the groups that helped establish neurovascular coupling as a measure that could be tested in humans. This involved kind of understanding metabolism of the eye, how that's coupled to the visual cortex, and how to measure blood flow on a high temporal resolution in the visual cortex in response to visual input. That's why we used very well standardized perturbations involving tracking an eye, tracking a dot on a screen at a known one rate and a known one amplitude of movement, while also measuring the hyperemic response in the posterior brain.   Then we kind of went on and developed some new measures, developed some software that we're now proud is used in a few different labs around the world, that kind of automatically takes that input of repetitive eyes opening and closing and that hyperemic response, and it breaks it down into a single wave form. A single hyperemic response is superimposed of 10, 15, 20 cycles of those eyes open and eyes closed, and then when we superimpose all the wave forms together, we can generate different metrics from that hyperemic response that correspond to different elements.   One of the ways where software can, I guess dice out the hyperemic response, is by timing. We can look at very specific unique time windows over that 30 seconds of eyes open, and we can also look at the slope of the response, as well as we recently did some dimensionality reduction techniques and looked at specific computed measures of that hyperemic response. We published that a few years ago. Those were some of the tools that enabled this study, along with a fantastically unique drug that really could isolate that neuron expression of NOS and the capacity of nNOS to mediate neurovascular coupling.   Kevin O'Gallagher:    Obviously, we're going to use a systemic infusion of SMTC, the study drug, and we've used that before and shown it to be safe. But because a systemic infusion of SMTC through peripheral and systemic nNOS inhibition does cause an increase in systemic vascular resistance, and therefore an increase in mean arterial pressure of around about 7 mm of mercury, in addition to a cline placebo control condition, we also felt the need to have a pressure control condition. For that, we used phenylephrine to match the rise in mean arterial pressure that we anticipated we'd see with SMTC. We ended up with 12 healthy volunteers who attended on three separate visits, and so we had a party randomized double blinded intervention study where we measured the neurovascular coupling metrics, both before and after an infusion of one of the three conditions on each particular visit.   Aaron Phillips:            I just wanted to add into that, we had found previously that mean arterial pressure does have an effect on the hyperemic response. This was actually classically found by 1960s by Harper and Glass in a dog study, but we've repeated that in humans and kind of found that the ability of the brain to kind of... It's reserve for further vasodilation is dependent on pressure. As you drop it, neurovascular coupling will go away, and as you increase it, neurovascular coupling will increase partially, so it's important to standardize the mean arterial pressure levels. I always liken it to your water pressure in your house. You can't turn on a faucet with a given pressure unless you have that in the system upstream. That was a really important aspect of the study.   Nicole Purcell:             That was quite unique for your study, too. Not a lot of people have control for pressure.   Aaron Phillips:            Correct.     Kevin O'Gallagher:    I think it reflects the challenges of these healthy volunteer studies where you're trying to look at one particular part of the cardiovascular system, because as a cardiologist, if we were doing a study like this, looking at cardiovascular regulation, we would put a catheter into the coronary arteries in patients who had come for angiograms, and we'd give a local infusion of SMTC, as we've done in studies before. But with healthy volunteers, and ethically it really demanded a systemic infusion, so it was a really nice workaround to have that pressure control condition.   Nicole Purcell:             So, can you tell us a little bit about what your findings were?   Kevin O'Gallagher:    I think testament to the study design and the rigorous methodology that we employed, we did find with the resting steady state hemodynamics that SMTC condition performed as we would expect, and as we've seen in prior studies where we've given a systemic dose in that compared to both placebo and pressure control conditions, SMTC decreased cardiac output, and it decreased stroke volume, and also increased systemic vascular resistance, so very much as expected the resting hemodynamic conditions.   Aaron Phillips:            Yeah, thanks. Just adding onto that, moving on into some of the cerebral vascular measures. So again, we were measuring posterior cerebral artery velocity, blood velocity and specific responsiveness that it has to a visual stimuli. Between conditions, we didn't see a change in resting posterior cerebral artery velocity, so that was consistent between the conditions. Where we saw most of our change actually was in this very early period, the first five seconds of what we're going to call the hyperemic response, or the first five seconds of the neurovascular coupling response. That's where we saw our primary effect. We didn't see an effect in almost any of the neurovascular coupling measures that we generated in the actual sustained period after that initial rise, so that's where we saw our key inhibition with nNOS inhibition. What permitted that was the phenylephrine control group, again, allowing us to really look at apples and apples, not apples and oranges.   Nicole Purcell:             Great. So that early transient change that you saw, that as you said, hyperemic response, what therapeutic implications does this have for the field?   Kevin O'Gallagher:    Well, certainly there are conditions in which nNOS dysfunction, nNOS may be implicated, we mentioned a couple in the paper, some neurodegenerative diseases. But also, I think the field is now open for any vascular mediated headache syndrome, such as migraine, to investigate the potential role of nNOS from that angle. Then we haven't touched on already, but as well as dysfunctional, so decreased nNOS activity, there's also some conditions in which there's dysregulation or abnormally increased nNOS function. Again, we've highlighted this kind of study methodology is a tool that could be used to investigate those types of conditions.   Aaron Phillips:            These are all terrific points, and I think there's a lot of conditions where neurovascular coupling is impaired, and it's worth exploring them and understanding the specific role where nNOS might be a part of it. I also think there's a lot of interesting basic science surrounding this, in terms of the mechanisms. What was really interesting in this study, which is still kind of wracking my brain, is why didn't more of the neurovascular coupling response go away? This is a highly selective inhibitor for what was potentially thought by some groups to be a large mediator, this response. It was a relatively small inhibitory effect, and isolated to a small part of the neurovascular coupling response, just that early phase. So, still lots of work to do to kind of dice out the other pathways. They're probably highly redundant. This is such a critical mechanism in the central nervous system. Getting at it and humans is going to be tricky, but we're excited about the future and exploring some of those other avenues on the mechanistic cascade.   Nicole Purcell:             Based on the fact that you just had 12 healthy individuals, what do you see as some of the limitations of your study going forward, thinking about what you did?   Kevin O'Gallagher:    I think you've just hit on a key limitation. It was a small number of volunteers. They were all healthy, so we can't extrapolate these findings to conditions such as hypertension, where we know from other studies that cardiovascular responses, nNOS responses are impaired Also, this was a noninvasive study. We looked at the blood flow through Doppler, but we don't really know the effect of SMTC on cerebral artery diameter or other markers like that, so I think those are important limitations to mention.   Nicole Purcell:             I know I didn't ask this, and I know it was mentioned in the paper, but for our audience, and it was a small sample size, but did you see any sex differences between your male and female cohort?   Kevin O'Gallagher:    No. We did analyze for that and there were no sex differences. But again, it's an important limitation in that we didn't control for things like phase of the menstrual cycle. And again, with those limitations, all the results should be interpreted with those in mind.   Nicole Purcell:             Were there any challenges to the study that you found?   Kevin O'Gallagher:    I work in London in the UK, where we performed this study related protocols, and Professor Phillips from University of Calgary, his team flew over to perform the studies. I think there was a real organizational challenge because we had a relatively small time window in which to get all of the volunteers and their three study visits done. But I think it's testament to just how well Professor Phillips runs his team, and how fantastic a team they are in working together that all of those challenges were minimized and everything. It ran fairly smoothly, and certainly, the data was connected back in early 2020. I think we all retrospectively breathed a sigh of relief when the Covid pandemic started and we realized that had we had to reschedule another set of visits, we would've then knocked the study back a couple of years. So yeah, there were organizational challenges, but it was an absolute pleasure to work with Professor Phillips's and his team in this.   Aaron Phillips:            To add to that, I mean, it's not really related to necessarily the challenges, but I was going to list kind of the exact same thing. In the background. Kevin, and Professor Shaw, and Dr Gallagher were a tour de force on organizing quite a complicated study that involves some invasive protocols and unique experimental drug infusion. Getting all of that ethically approved, and organized, and structured, that was probably one of the biggest challenges of pulling this study off. Nicole Purcell:            Great. It was a very nice study. So lastly, what future studies are needed or have come out of this work that you'd like to tell us about?   Aaron Phillips:            Mechanistically, I would still like to explore why nNOS inhibition doesn't seem to affect the sustained elevation in blood flow. This maybe means going back to some of the astrocyte mediated mechanisms, and understanding knocking out, knocking in, exploring some of those. I'd also like to continue to study the neurovascular cupping response itself in clinical conditions. This may be a tool for helping to characterize the severity of a given neurovascular condition over time, and kind of validating this outcome measure as potentially a clinical tool and further expanding its research application.   Kevin O'Gallagher:    I would just add to that, that I tend to come to all of these things from a cardiologist light, and there are some conditions in cardiology where the microvascular is involved, and so the interest is then to see whether there's a linkage between the dysfunctional coronary microvascular responses with then cerebral microvascular responses. So again, I think there's plenty of future work to be done in that sphere.   Nicole Purcell:             Well, I want to thank you so much for joining me today, Dr Kevin O'Gallagher and Dr Aaron Phillips, for discussing your exciting findings with me today, and I look forward to seeing your future work. Thank you.   Aaron Phillips:            Thank you so much.   Kevin O'Gallagher:    Thank you so much.   Nicole Purcell:            That's it for highlights from the December 2 issue of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Aaron Phillips and Kevin O'Gallagher. This podcast is produced by Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles provided by Ruth Williams.   I am your host, Dr Nicole Purcell, filling in for Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more, visit ahajournals.org.