March 2020 Discover CircRes

 

This month on Episode 10 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the February 28 and March 13, 2020 issues of Circulation Research and talks with Dr Mary McDermott about her article Cocoa to Improve Walking Performance in Older People With Peripheral Artery Disease: The Cocoa-Pad Pilot Randomized Clinical Trial.

 

Article highlights:

 

Rykaczewska, et al. PCSK6 Is a Key Protease in Vascular Injury

 

Lebek, et al. SDB Induces Arrhythmias via CaMKII and Late Ina

 

Mueller, et al. Brain Damage With Heart Failure

Napierski, et al. Cut and Paste of cMyBP-C Domains In Situ

Transcript

Cindy St. Hilaire: Hi. Welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm from the Vascular Medicine Institute at the University of Pittsburgh.

Today I'm going to share with you four articles selected from the February 28th and the March 13th issues of Circulation Research as well as have an in-depth discussion with Dr Mary McDermott, who is the corresponding author of the study COCOA-PAD Pilot Randomized Clinical Trial. So first, the highlights.

The first article I'm sharing with you is titled PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling. The first authors are Urszula Rykaczewska, Bianca Suur, Samuel Röhl, and the corresponding author is Ljubica Matic from the Karolinska Institute in Stockholm, Sweden.

The family of proprotein convertase subtilisins/kexins, or PCSKs case for short, are a group of proteases whose role in vascular disease was only recently recognized. Humans with gain- and loss-of-function mutations in PCSK9 exhibit very high or very low levels of cholesterol, respectively, and this information was leveraged for the development of novel, albeit extremely expensive, drugs for regulating cholesterol. However, the role of other members of the PCSK family in cardiovascular disease is not known.

This group previously found that PCSK6 was one of the most enriched molecules in human carotid artery plaques as compared to normal arteries, while other PCSK family members did not show the same trend. This prompted the group to further explore the role of PCSK6 in vascular disease. They used a very integrative approach drawing from several independent human biobanks for genetic information, conducting in situ functional investigations using human tissue, also conducting in vivo animal models of vascular injury, including using the PCSK6 knockout mice, as well as ex vivo and in vitro mechanistic studies.

And they found that PCSK6 was a key modulator of smooth muscle cell function in vascular remodeling and atherosclerosis through a very novel mechanism implicating MMP14 and MMP2 activation upon cytokine stimulation. Future studies will investigate the role of PCSK6 on atherosclerotic plaque remodeling and instability because, as we know, plaque rupture can have devastating consequences.

The second article I will highlight is titled Enhanced CaMKII-Dependent Late I Na Induces Atrial Pro-Arrhythmic Activity in Patients with Sleep-Disordered Breathing. The first author is Simon Lebek, and the corresponding author is Stefan Wagner, from the University Hospital Regensburg in Regensburg, Germany.

Sleep-disordered breathing is an umbrella term for any chronic condition involving the complete or partial interruption of breathing during sleep, and this is commonly called sleep apnea. Aside from daytime sleepiness, people with sleep-disordered breathing run the risk of developing arrhythmia, such as atrial fibrillation.

Arrhythmias are an electrical problem as opposed to a mechanical one. And at the cellular level, arrhythmias are associated with increased activity of the enzyme calcium/calmodulin-dependent protein kinase 2, or CaMKII, and this protein regulates cellular electrophysiology. Despite the role of CaMKII in propagating electrical signals in the heart, its activity has not been investigated in sleep-disordered breathing patients.

This group now shows in a study that used 113 patients undergoing heart surgery that those with sleep-disordered breathing have higher CaMKII in biopsied myocardium than those without the condition. Furthermore, this CaMKII increase was associated with other pro-arrhythmic alterations to the tissue, including increased reactive oxygen species production, enhanced phosphorylation of a major sodium channel, and consequent late firing of sodium currents.

Importantly, these alterations could be prevented by pharmacological inhibition of CaMKII, suggesting that such an inhibitor could be a novel treatment strategy for patients with sleep-disordered breathing to reduce their arrhythmia risk.

The next article I want to share with you is titled Brain Damage with Heart Failure: Cardiac Biomarker Alterations and Gray Matter Decline. The first authors are Karsten Mueller and Friederike Thiel, and the corresponding author is Matthias Schroeter, and the work was completed at the Max Planck Institute for Human Cognitive and Brain Sciences in Leipzig, Germany.

Heart failure leads to decreased blood flow due to a reduced pumping efficiency of the heart, and as a consequence, this can cause insufficient oxygen supply to the tissues, including the brain. Cardiovascular insults, including heart failure, increase the risk for the development of neurological diseases later in life, such as vascular dementia and Alzheimer's disease. Patients with heart failure can show neurological symptoms such as fatigue, nausea, and dizziness. However, the long-term consequences of the effects of heart failure on brain integrity are not well understood.

However, several studies suggest that structural changes in the gray matter can occur. This study sought to identify correlations between cardiovascular biomarkers and structural gray matter changes in the brain. They found that patients who suffered from heart failure undergo detrimental brain structural changes. Reduced gray matter density in several regions of the brain correlated with decreased ejection fraction at baseline and increased NT-proBNP, which is a heart failure biomarker. While these observations might reflect structural brain damage in areas that are related to cognition, whether these structural changes facilitate the development of cognitive alterations will need to be proven in future longitudinal studies.

The last article I want to share with you before we switch to our interview is titled A Novel "Cut And Paste" Method for In Situ Replacement of Cardiac Myosin Binding Protein C Reveals a New Role for This Protein in the Regulation of Contractile Oscillations. The first author is Nathaniel Napierski, and the corresponding author is Samantha Harris, and they're from the University of Arizona.

Actin and myosin are the respective thin and thick filament proteins that allow for muscle contraction, including in the cardiomyocytes, the muscle cells of the heart. Cardiac myosin binding protein C is a critical protein that regulates heart contraction, but the mechanisms by which this protein affects actin and myosin are only partially understood. One reason for this is that cardiac myosin binding protein C localization on the thick filaments may be a key component of contraction, but most in vitro studies cannot spatially replicate arrangements of cardiac myosin binding protein C within the sarcomere.

To address this technical gap, this group created a novel hybrid genetic/protein engineering approach that allows for rapid manipulation of cardiac myosin binding protein C in sarcomeres of permeabilized myocytes isolated from genetically engineered Spy-C mice in situ. So essentially, they can do some gene editing in tissue in situ. Using this approach, they were able to rapidly remove and replace cardiac myosin binding protein C. Deletion of this protein fully recapitulates effects obtained using traditional knockout and transgenic mouse models of cardiac myosin binding protein C.

However, the ability to rapidly remove and replace this protein identified a new regulatory role for cardiac myosin binding protein C where it functions to dampen contractile oscillations. The novel cut and paste approach should be very useful in testing these new hypotheses of the role of cardiac myosin binding protein C function as well as in defining the role of how spontaneous contractile oscillations affect cardiac contractility during both health and disease.

Okay. Now we're going to switch over to the interview portion of the podcast. I have with me Dr Mary McDermott from the Departments of Medicine and Preventative Medicine at Northwestern University's Feinberg School of Medicine. And today we're going to be discussing her manuscript titled Cocoa to Prevent Walking Performance in Older People With Peripheral Artery Disease: The COCOA-PAD Pilot Randomized Clinical Trial. Thank you for joining me.

Mary McDermott: Oh, it's a pleasure to be here.

Cindy St. Hilaire: Before we dig into the details and the nitty-gritty of the study, could you maybe first explain to us what peripheral artery disease is and perhaps maybe why it's so pernicious?

Mary McDermott: Sure. So peripheral artery disease is atherosclerosis of the arteries that supply the legs, and it is a problem because it causes great difficulty with walking. People with peripheral artery disease, or PAD, can typically walk only at one or two blocks before they have to stop because of symptoms or weakness or pain or tightness in their lower extremities, in their legs. And it's also difficult to treat because we have very few medical therapies available that are effective.

Cindy St. Hilaire: So this is really something that you only know it's there until you're feeling the adverse symptoms.

Mary McDermott: That's correct.

Cindy St. Hilaire: That sounds very difficult to treat clinically. This study was called the COCOA-PAD study, and it was a double-blind pilot, randomized clinical trial, and it was designed to test the hypothesis that daily cocoa consumption for about six months improves or prevents the decline in something called the six-minute walk distance test. So my first burning question is, is it okay to eat a lot of chocolate every day? And then my second question is, what is the significance of the six-minute walk test? And maybe you could tell us a little bit about this trial's design.

Mary McDermott: Sure. Maybe I'll go in reverse order for those questions.

Cindy St. Hilaire: Sure.

Mary McDermott: So the trial design, it was a randomized clinical trial. 44 participants with peripheral artery disease were randomized to receive either the cocoa beverages, we asked them to take three a day, or a placebo control, which was very much like the intervention except that the placebo did not have cocoa or cocoa flavanols. And participants were followed for six months, and at baseline and six-month follow-up, we measured the six-minute walk test, and we also did muscle biopsy on those who consented to that, and we also measured lower extremity perfusion with MRI.

Now, the six-minute walk test is a test that's very well-validated in patients with peripheral artery disease, and it's really a measure of walking endurance. The way you conduct it is you need a hundred-foot hallway. We use standardized instructions. We actually use a script where the research assistant reads the script with the instructions, and the goal of the test is for the participant to walk as many lengths as they can in the six minutes. And often what you see in people with peripheral artery disease is they start out fine, but after maybe a few hundred-foot lengths, they start to slow down or they start to limp, and many of them cannot finish the six-minute walk without having to stop and rest. If they need to stop, then they can start walking again.

Cindy St. Hilaire: That's interesting. Is it a fatigue or is it a pain, or what is prompting them to stop?

Mary McDermott: It's symptoms in the legs or the hips classically, and it can be either of those symptoms that you mentioned. It may be a fatigue or a weakness. Some people will say, "I don't have pain. My legs just get weak, and I can't keep walking." Others will have pain. Many will have tightness or burning. And it's interesting, some people will get symptoms mainly in their feet or ankles, others will get it classically in the calves, but many will get it in the hips. And the location depends in part on the location of the atherosclerosis and where they're experiencing the ischemia.

Cindy St. Hilaire: Interesting. And so ultimately this is due to the atherosclerotic plaque blocking blood flow?

Mary McDermott: Exactly. Right. So when they go to walk, their muscles are not getting an adequate oxygen supply, and that causes these symptoms or weakness in the legs.

Cindy St. Hilaire: Interesting. What's so special about cocoa, and what are these flavonoids that you mentioned?

Mary McDermott: Sure. Cocoa actually comes from the cacao plant, and in that plant there's cocoa, but also something called cocoa flavonoids, and this is a nutritional substance. And there's a variety, but in cocoa, epicatechin is the most prevalent flavonoid.

And flavonoids have health benefits that include improving blood flow by causing dilation of vessels, but also, they've been shown to have favorable effects on muscles, skeletal muscle. And so this is particularly potentially helpful in peripheral artery disease because, obviously, patients with PAD have difficulty with blood flow because of those atherosclerotic blockages, but also they've been shown to have skeletal muscle abnormalities, probably because their leg muscles aren't getting enough oxygen. So they develop loss of muscle mass, they develop mitochondrial dysfunction and other abnormalities in their muscle that also make it hard for them to walk. So cocoa and cocoa flavonoids are an attractive therapy in PAD because they both can improve blood flow and improve the health of the skeletal muscle in the legs.

Cindy St. Hilaire: Interesting. So it's kind of a two-pronged approach to possibly helping these patients. What was the scientific evidence out there that the flavonoids or maybe even just dark chocolate may be beneficial, and how was your study different from other studies?

Mary McDermott: Some of the evidence comes from animal studies where it's clearly been shown to improve skeletal muscle mitochondrial activity and muscle growth and also blood flow. But there were also some preliminary studies in humans, a couple of them really small sample sizes of patients with heart failure, showing improvements in skeletal muscle health. But there was one trial published about five years ago in patients with PAD where the PAD patients were given one dose of dark chocolate or one dose of milk chocolate, and that one dose helped them achieve increased walking distance on a treadmill about two to three hours later. But to our knowledge, no prior studies had tested whether a daily dose of cocoa could improve six-minute walk or improve skeletal muscle or blood flow.

Cindy St. Hilaire: So should I eat chocolate every day?

Mary McDermott: Well, there's a couple of important things about chocolate. First of all, most of the chocolate that you can buy at the store is not the type we used in our study. Oftentimes chocolate is alkalized, and what that does is it makes it taste better, but it also removes some of those cocoa flavonoids that are thought to be responsible for the health benefits.

The cocoa that we studied was rich in the cocoa flavanols. It had not been alkalized, and it was more of the dark chocolate. So if you want to eat it for health benefits, you need to read the label, and it should tell you whether the chocolate has been alkalized.

The other thing to take note of is, of course, many forms of chocolate come with a lot of calories or sugars, so that can be problematic if it leads to weight gain. The chocolate that we used in our study and the placebo added about 180 calories per day to the diet, and prior to starting the study we did a little bit of diet counseling with all the participants, and we helped them identify drinks or foods they were eating that maybe could be removed so that they could take the 180 calories without gaining weight. And we did not find weight gain in either group in this study.

Cindy St. Hilaire: That's good. That's good. One of your results I found interesting was that it showed that this daily supplementation of cocoa in the diet improved the six-minute walk test at a timeframe that was shortly after the chocolate dosing, but not 24 hours after. Can you maybe talk about that result and what the implications for that mean?

Mary McDermott: Sure. Because of that prior trial that I mentioned, which indicated that cocoa had an acute effect, we were interested in separating out the acute and the chronic effects. So we did two six-minute walk tests at six-month follow-up. The first was performed two and a half hours after the final cocoa dose, and the second was performed 24 hours later.

And we saw the biggest benefit at the time point that was two and a half hours after the final cocoa dose. The benefit was about 42 meters favoring the cocoa intervention. When participants came back 24 hours later, the difference between the intervention and the placebo was only 18 meters, and that didn't quite achieve statistical significance in our primary analyses.

Now, we were a little surprised by the difference in those findings. One possible explanation is that cocoa has both the chronic and acute benefit and that first measurement reflected both the acute and the chronic benefits. So that's one possible explanation.

Another is, interestingly, we found that the placebo group had a bit of a learning effect between the two-and-a-half-hour time point and the 24-hour time point, and it's possible that that explained the diminishment in the difference of the 24-hour time point. But we didn't see that learning effect in the cocoa group, so that didn't quite make sense.

In my mind, the best explanation is there may be both an acute and a chronic effect, and we saw the benefit of both of those at that first time point.

Cindy St. Hilaire: Interesting. And a learning effect, by that you just mean the patients just learned to do the test better?

Mary McDermott: Yeah. So they got more comfortable with it between the first and the second measurement, which were just 24 hours apart. Prior study in peripheral artery disease patients has not shown a learning effect. But in the prior study, the six-minute walk tests were performed one or two weeks apart. And to my knowledge, no one's ever tested it just 24 hours apart.

Cindy St. Hilaire: Interesting. Very interesting. What was really the most challenging aspect of  this study? Can you talk about some of the limitations also?

Mary McDermott: Sure. I'd say the biggest limitation was the sample size. This was a pilot study. It's not a definitive result. There were 44 people, so that is certainly a limitation. And perhaps related to that, we did see some imbalances at baseline between the two groups in terms of BMI and prevalence of African-Americans between the two groups. Our analyses do adjust for those differences to try to overcome that potential difference.

With regard to challenges, well, recruiting for studies of peripheral artery disease is always a challenge because the patients are limited in their own mobility, and it can be hard for them to come in for the study visits. A study like this requires multiple visits at baseline and follow-up.

Another potential challenge is that the adherence rate was about 64% in the intervention group versus closer to 80% in the placebo group. We don't know-

Cindy St. Hilaire: Oh, interesting. What do you think that is?

Mary McDermott: We don't know exactly why. It's possible that the cocoa intervention had a different taste and maybe was not as palatable, but since participants only had their own drink, we didn't ask them to compare, and we can't say that for sure. That could've been just due to chance.

Cindy St. Hilaire: Sure. Wow. Well, hopefully, a future study can help figure that out. Speaking of that, what would be next really in terms of kind of translating this study into either a bigger study or really translating it to the clinic? What do you see for this moving forward?

Mary McDermott: Well, couple things. I think most immediately, because there are so few therapies for peripheral artery disease and because cocoa has essentially no side effects with the caveat being the potential for weight gain, that it would be reasonable to recommend it to patients who are really symptomatic and can't seem to get better with standard options such as exercise or maybe in addition to exercise.

But I do think before we can reach a definitive conclusion, a definitive trial is needed. We have applied, we have submitted a grant application to obtain funding to do a larger study, but we'll need to wait and see how that goes.

Cindy St. Hilaire: Well, hopefully, that gets funded because I would love any excuse to eat a little more chocolate, even if it's non-alkalized. Well, great. Well, thank you so much for joining me today, Dr McDermott. This is a wonderful study, and I wish you the best of luck on that next funding to do a larger study.

Mary McDermott: Well, thank you so much. I really appreciate your interest in this work. Thank you.

Cindy St. Hilaire: That's it for highlights from the February 28th and March 13th issues of Circulation Research. Thank you so much for listening.

This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. Thank you to our guest, Dr Mary McDermott. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.