Melanie Cole, MS (Host): Epidemiological and genetic studies have shown that disruption of the circadian clock is a factor in multiple pathologies, including metabolic disease, myopathy, and cancer. Welcome to Better Edge, a Northwestern Medicine Podcast for physicians. I'm Melanie Cole.

And joining me today is Dr. Clara Peek. She's an Assistant Professor of Biochemistry, Molecular Genetics and Medicine in the Division of Endocrinology at Northwestern Medicine. Dr. Peek is here today to walk us through a new Northwestern Medicine study that she led that was published in Science Advances, suggesting that circadian rhythms play a role in how quickly damaged muscles heal.

Dr. Peek, thank you so much for joining us today. And can you start by explaining the current understanding of the circadian clock in stem cell function, particularly in muscle repair and regeneration? Explain to us a little bit about the genes that form a molecular circadian clock that could help regulate many body functions.

Clara Peek, PhD: Yeah. Thanks for having me. So, our lab studies, these proteins that are expressed from the genes that comprise our molecular circadian clocks, and these protein factors, they drive our body's circadian rhythms. So, these are our 24-hour cycles of things that we think of as being typically circadian like behaviors like sleep and feeding, but it also controls virtually all other processes in our bodies like body temperature, hormone secretion, and even intracellular metabolism. So, we focus on the circadian clock within skeletal muscle tissues.

And a few years ago, we actually found that, within the stem cell populations in our muscles, these are also called satellite cells. They're actually essential for muscle tissue regeneration following things like exercise or injury. So, they're kind of how we repair our muscles and also how we build muscles when we exercise. We found that they also express this circadian clock network. So, these proteins are present in stem cells and they're rhythmically expressed. So, the clock is ticking in these cells. This suggested that there was circadian regulation of stem cell function.

And in our previous work, we also found that this stem cell clock actually drives day-to-night rhythms in our capacity of our muscles to regenerate themselves after an injury. But at that point, we didn't actually know how this was happening on the mechanistic level. And so, in the current study, we investigated that mechanism.

Melanie Cole, MS: This is so interesting. I'm an exercise physiologist. And so, I'm interested in how muscles heal from injuries. And it's really a big field. So Dr. Peek, give us a little overview of the study, your primary research objectives. And what we had learned and what you were just saying, is that mice healed faster when injured during their active hours compared to their sleep hours. How does the current study aim to explore how these circadian clocks in muscle stem cells influence that regeneration at different times of the day?

Clara Peek, PhD: Yeah. So, using a variety of methods including conditional genetic models where we can actually just lose the clock, specifically within the stem cells in adult life in mouse models, and then also using technology called single-cell RNA sequencing of muscle tissue both before and after injury at various times of day.

What we found was that these circadian clocks within the muscle stem cells drive the metabolic responses to injury. And so after injury, the muscle becomes actually oxygen-limiting. So, there's a metabolic stress that happens. And so, we found that the clock is timing the way in which the cell adapts to this by shifting from using oxygen as sort of its electron carrier and ATP-producing mechanism in the mitochondria to anaerobic glycolysis. And what was really interesting using single-cell sequencing is that we also simultaneously found that the muscle stem cell clock was also timing inflammatory responses. And we were able to actually show that these things are connected. So, this metabolic signal driven by the clock in the stem cell is actually important for this inflammatory signaling after injury that is actually important to recruit neutrophils, which are known as like the first responder innate immune cells following injury. And they're actually very important for clearing injured muscle debris and also recruiting other cell types and even driving muscle stem cell proliferation itself to efficiently repair the muscle.

So, we found that this signaling between the stem cell and the neutrophil was stronger right after injury when the mice were injured during their wake periods. So, mice are nocturnal, this was during the nighttime. And then, we further found that when you lose the stem cell clock using these conditional mice, muscle regenerative capacity was constituently impaired. So, it was poor, but it was poor at all times of day, and there was no rhythm of regenerative capacity. So, this really showed that a healthy clock is required in the stem cell for maximal muscle repair and regeneration.

Melanie Cole, MS: What made this approach useful for uncovering that time of day effects?

Clara Peek, PhD: Well, so the conditional genetic models are obviously very valuable. They've been around for a while. We also teamed up with Navdeep Chandel, also at Northwestern, who had these mice that actually express an NAD, which is a co-factor that is important. That is the signaling metabolite produced by anaerobic glycolysis. And we could actually just express this enzyme specifically in muscle stem cells. So, that really allowed us to assess this sort of causal relationship between the glycolytic effect and the innate immune signaling. And then, also the use of single-cell technology really enabled us to ask this question whether or not the stem cell clock is mediating interactions with other cell types.

So, the molecular clock is expressed throughout the body, as I mentioned, but a question has remained as to whether these cell autonomous clocks are acting alone to control their own biology or whether they're interacting with other cell types to control rhythms of complex cell-cell interactions and processes like development.

So now, with the advent of single-cell sequencing, we have the capability to study the coordinated responses and interactions between cell types in this case and injured muscle tissues at different times over the 24-hour cycle. So, it's a really exciting time in frontier.

Melanie Cole, MS: It certainly is. And for context, Dr. Peek, can you explain the transcription-translation feedback loop that underlies the circadian clock's function and how it may impact gene expression related to muscle repair?

Clara Peek, PhD: Yeah. So, the mammalian molecular circadian clock is made up of transcription factors. So, there are activators, so these are proteins that turn on genes, and there are also repressors that are part of this clock network. And what they do is they form a negative feedback loop whereby the activators control the expression of their own repressors in a cycle that repeats itself every 24 hours. And these factors, as I mentioned, are expressed in various cell types. And a lot of studies now are focused on understanding the full transcriptome, so all the genes regulated by these. So, they regulate themselves to keep the cycle going, but they also regulate many different types of cells in various pathways and tissue-specific pathways, because it turns out that the clock regulates a unique transcriptional program in each cell type, including muscle stem cells. And so, we found that the major clock-regulated pathways in muscle stem cells following injury included those involved in anaerobic glucose metabolism, including the production of this co-factor, NAD, and also inflammatory responses. So, that kind of gave us the clue that maybe these things are connected.

Melanie Cole, MS: We'll expand a little bit then for us, Dr. Peek, on NAD. Your study highlights the role of it in regeneration in the muscle stem cells. What are the potential implications of this finding for understanding muscle repair in clinical settings, especially in patients with metabolic disorders?

Clara Peek, PhD: Yeah. So as I mentioned, we found that the stem cell clock dictates time of day differences in neutrophil activity through regulation of this stem cell metabolic response to injury. And we found that the regeneration of this anaerobic glycolytic product, nicotinamide adenine dinucleotide, or NAD, is important for this process.

And so, NAD is made in our bodies in a number of ways, including from the dietary vitamin B3. It's a critical redox factor, and it's important for many, many biochemical reactions in our body. But also, it acts as an important signaling metabolite that is known now to influence transcriptional regulators. So, these include these factors called PARPs and also sirtuins. And these can be very important for adaptation to different conditions of metabolic stress. They've been implicated in cancer. And so, there have been now preclinical and clinical studies that point towards a beneficial role of B3-like supplements, which are basically precursors to boost NAD production in our cells.

People have looked at this in conditions like aging insulin resistance, and also in muscle pathologies, like for example, vascular ischemia. And so, another important thing to note is that, in addition to being produced by the clock, a number of previous studies-- not by us-- but have established that there's a reciprocal regulation of the circadian clock by NAD as well.

So, NAD seems to be bidirectionally linked to circadian function. Because of that, I think an important next question is whether or not these beneficial effects of NAD supplementation, taking these B3 precursors are imparted by improving the clock or its downstream pathways.

Melanie Cole, MS: Wow. Fascinating the implications. And while we're thinking of this, I'd like you to take us from bench to bedside as these findings could be significant for studying how disruptions in circadian rhythms that we often see in aging, obesity, how they affect muscle healing. How does aging or disease, when we think of the population, affect that circadian regulation of muscle stem cells? The implications for this, for therapeutic strategies, where do you see it playing the biggest role in our aging community?

Clara Peek, PhD: So as you mentioned, there is a connection between conditions like metabolic disease, like metabolic syndrome, insulin resistance, aging, and circadian rhythms. So, it's kind of also this bidirectional relationship where in those conditions actually the transcriptional program of the circadian clock is altered or reprogrammed. And this has actually been shown in muscle stem cells in another study. It's known that the circadian genes are reprogrammed to control totally new processes that are not controlled in young mice. So for example, oxidative stress and inflammation.

And then, reciprocally, it's important to note that in those same states, like aging and metabolic diseases like diet-induced obesity, there's also reduced circadian rhythmicity. So specifically, rhythms are known to be shifted a bit and also generally dampened in amplitude. This has been shown now and is well established in both animal models and in humans. So really, where this leaves us is I think a vital question in the field of circadian rhythms in general and also in our research is the degree to which impaired circadian function underlies some of these negative pathologies associated with these conditions and aging in general.

And so, on our end, we've done some preliminary studies now where we have observed that time of day differences in muscle regenerative capacity are actually lost in mice that are given a diet, like a western diet to induce diet-induced obesity and insulin resistance. So, this suggests that actually their clock is impaired in the whole animal, but also in the muscle stem cell itself. So, this is a really kind of the new area of investigation where we're taking this research.

Melanie Cole, MS: Fascinating. And considering the correlation between disrupted circadian rhythms as you've been talking about here today, and impaired muscle regeneration that we've mentioned in obesity, aging, metabolic disorders, in what way do you see the interactions between neutrophils and muscle stem cells altering the therapeutic approaches to muscle injuries in a clinical environment? And importantly, how this could inform prevention or treatment strategies in these populations, and a great word being prevention?

Clara Peek, PhD: Yeah, our findings suggest for the first time that the stem cell clock pathway is an important regulator of muscle injury repair, which involves inflammatory responses. And it ultimately dictates the efficiency of muscle repair. So like, what are the implications, I think, is that a next big question would be to determine whether targeting the circadian clock factors themselves, there are drugs that modulate the clock or maybe even boosting some of these downstream clock-regulated pathways like NAD or even some of these innate signaling cytokines right after injury, could those actually enhance muscle regenerative capacity in populations with reduced circadian function, like older individuals that are suffering from age-related muscle pathologies, like sarcopenia or vascular ischemia?

But then, I think, to your point, I think prevention is actually where a lot of this could be important as well in terms of maintaining a healthy circadian rhythm within our stem cells, either through supplementation of NAD, which are already readily available, or even just behavioral changes, lifestyle changes, like things like intermittent fasting or time-restricted feeding and exercise have actually been shown to boost healthy circadian rhythmicity.

Melanie Cole, MS: Wow. This has been an absolutely enlightening discussion. As we wrap up, looking forward, what do you envision as the future direction for research or potential therapeutic interventions targeting the circadian clock in muscle stem cells? Where do you see it going? Where would you like it to go? Or where would you like to see it go? And what would you like other providers to take away as the key messages from this discussion today?

Clara Peek, PhD: I think from just a research perspective and also just an understanding of the importance and influence of circadian biology and disease and injury would be to just really understand what is the causal role of circadian disruption in conditions like aging and metabolic disease on decline of functions like regenerative capacity, for example. And that could even be in response to injury, but even in some of these states that are aging-related, like vascular ischemia, I think applying these to more translational models like limb ischemia, for example, is a really exciting new area that I think if we show that circadian disruption is associated with this condition, but also that it can actually exacerbate the pathologies associated, like the impaired regenerative capacity and muscle maintenance. It is really kind of the next big step to sort of investigate whether or not this is a translational and causal factor underlying these disease states.

Melanie Cole, MS: Dr. Peek, I hope you'll join us again and update us as you learn more and these studies continue. That was such a great discussion, so informative. Thank you again for joining us. And to refer your patient or for more information, please visit our website at breakthroughsforphysicians.nm.org/endocrinology to get connected with one of our providers. That concludes this episode of Better Edge, a Northwestern Medicine podcast for physicians. I'm Melanie Cole.