Circulation on the Run

Each monthly episode will discuss recent publications in the fields of genomics and precision medicine of cardiovascular disease.
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Now displaying: September, 2018
Sep 24, 2018

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore.

                                                Ticagrelor has shown superior efficacy to clopidogrel in the management of acute coronary syndromes. But what about in patients undergoing PCI for stable coronary artery disease? Well, our feature paper this week gives us answers to this question but you're going to have to wait to the feature discussion to hear these answers. That's coming up right after these summaries.

                                                Our first original paper this week shows that RBM20 mutation carriers have an increased risk of arrhythmias. You may recognize RBM20 as that splicing factor which targets multiple pivotal cardiac genes such as Titin and Calcium/Calmodulin-Dependent Kinase 2 Delta or CAMK2D. In today's paper first author Dr van den Hoogenhof and co-corresponding authors Dr Pinto and Creemers from Academic Medical Center Amsterdam, compared the clinical characteristics of RBM20 and Titin mutation carriers and used RBM20 knock out mice to investigate the downstream effects of RBM20 dependent splicing. They showed that loss of RBM20 disturbed calcium handling and led to more pro-arrhythmic calcium releases from the sarcoplasmic reticulum. Patients that carried a pathogenic RBM20 mutation had more ventricular arrhythmias despite a similarly depressed left ventricular function compared to patients with a Titin mutation.

                                                Targets of RBM20 splicing were enriched for calcium and ion handling genes, most notably CAMK2D and type 2 Ryanodine receptor. Loss of RMB20 induced an increased L-Type Calcium current density, intracellular calcium overload, increased sarcoplasmic reticulum calcium content and increased spontaneous calcium releases which all could be attenuated with treatment with an L-type calcium channel blocker. Furthermore, these results suggest that RBM20 mutation carriers should be closely monitored for potential electrical disturbances and cardiac arrhythmias even in the early stages of disease.

                                                Echocardiographic quantitation of degenerated mitral regurgitation is recommended in clinical guidelines but is it really scalable to routine clinical practice? First author Antoine, corresponding author Sorano from Mayo Clinic Rochester Minnesota and their colleagues looked at more than 3900 patients diagnosed with isolated mitral valve prolapse between 2003 and 2011 and to any degree of mitral regurgitation quantified by any physician or sonographer in routine clinical practice. They found that in multi-variable analysis routinely measured effective regurgitant orifice area was associated with mortality independent of left ventricular ejection fraction and systolic diameter symptoms or age and comorbidities. Furthermore, compared with general population mortality excess mortality appeared for moderate mitral regurgitation with an effective regurgitant orifice area above 20 squared millimeters and became notable with an effective regurgitant orifice area above 30 squared millimeters which then steadily increased with even higher levels of above 40. Thus, quantitation of degenerative mitral regurgitation is scalable to routine clinical practice with strong independent prognostic power when performed routinely by multiple practitioners.

                                                The next study identifies a novel mechanism of lipid homeostasis that is linked to a pseudo gene associated with the recently discovered apolipoprotein known as APOO. Co-first authors Montasser and O'Hare, corresponding author Dr Mitchell from University of Maryland School of Medicine in Baltimore, performed an array based association analysis in more than 1100 Amish subjects and identified a variant strongly associated with LDL cholesterol levels. They identified a founder haplotype on chromosome 5 which was associated with a 15 mg/dl increase in LDL cholesterol after recombination mapping, the associated region contained eight candidate genes. Using a zebra fish model to evaluate the relevance of these genes to cholesterol metabolism they found that the expression of the transcribed pseudo gene APOOP1 increased LDL cholesterol and vascular plaque formation. Thus, based on these data the authors proposed that APOOP1  regulates levels of LDL cholesterol in humans and represents a novel mechanism of lipid homeostasis.

                                                The Orion-1 trial demonstrated that inclisiran which is a small interfering RNA therapeutic that targets PCSK9 MRNA with [inaudible 00:05:42] produces significant LDL reduction. In today's study from Dr Ray from Imperial College London and colleagues, the authors described in detail the effect of inclisiran on prespecified secondary lipid and lipoprotein outcomes over time for up to 210 days and also described the individual variation and response in these measures. They found that a single 300 milligram dose of inclisiran  lowered non-HDL cholesterol at day 180 by 35% and a second dose at day 90 resulted in a 46% reduction at day 180. Similarly a single dose of 300 milligrams of inclisiran  reduced apolipoprotein B by 31% at day 180 and a second dose of 300 milligrams administered in day 90 reduced apolipoprotein B by 41%. Significant reductions in all atherogenic lipoproteins measured were sustained through today 210. Furthermore, every individual had a reduction of apolipoprotein B and non-HDL cholesterol at 180 days with the 300 milligram two-dose regimen of inclisiran. Thus, inhibiting the synthesis of PCSK9 through small interfering RNA may be a viable alternative to monoclonal antibodies with respect to effects on atherogenic lipoproteins and that brings us to the end of our summaries. Now for our feature discussion.

                                                Ticagrelor has superior efficacy to clopidogrel in the management of acute coronary syndrome but it has not really been assessed in patients undergoing PCI for stable coronary artery disease. For our feature paper today it's going to shed some light and help us with this question and these are the results of the STEEL-PC trial. I'm so pleased to have with me right now the corresponding author Dr Robert Storey from University of Sheffield in the UK as well as our associate editor who managed this none other than Dr Stefan James from Uppsala University. Thank you.

                                                Rob, could you tell us what is the issue you tried to address and because your study is not that simple, we're not used to thinking about these pharmacodynamic and kinetic studies so could you explain a bit of what you did?

Rob Storey:                        Well it's quite a few concepts that we assessed in this study. We've got data from a number of studies showing that Ticagrelor both at doses of 90 mg twice daily and 60 mg twice daily is more reliable and superior P2Y12 inhibitor compared to clopidogrel. We've got this issue of very variable response to clopidogrel with some poor responders and some high responders and a range in between. That's fairly well established and part of this study was to get more data on the 60 mg dose of Ticagrelor in these stable CAD patients undergoing PCI and get some pilot data on clinical efficacy obviously this study was not part of clinical outcomes.

                                                But, there's another issue in terms of adenosine uptake so Ticagrelor has a relatively weak effect on adenosine uptake into red cells and other cells and this may or may not explain some of its clinical effects including some adverse effects such as dyspnea. We wanted to get a better idea of the impact of Ticagrelor at both these doses on adenosine uptake.

Dr Carolyn Lam:                Could I ask ... Okay this may be naïve. I'm not an interventional cardiologist but why would you expect something different in an acute coronary syndrome compared to stable coronary artery disease? Is there an underlying hypothesis there?

Rob Storey:                        Well there can be changes to their differences in platelet reactivity although those aren't particularly great and overwhelmed really by P2Y12 inhibitor like Ticagrelor which gives such reliable inhibition of the P2Y12 receptor. But, there have been a limited number of groups that have looked at adenosine uptake and so we wanted to get independent confirmation or not of whether Ticagrelor therapeutic concentrations impacting on adenosine uptake and get some ideas of whether it's affecting circulating adenosine levels. That's an important question in terms of understanding the mechanisms and actions of Ticagrelor.

Dr Carolyn Lam:                Got it. Thanks for breaking it down so nicely. So what did you find?

Rob Storey:                        What we found was surprisingly that we saw no impact of Ticagrelor at either dose and at any time point within a month after PCI on adenosine uptake. That is the circulating levels of adenosine and the rate at which adenosine is taken up by cells in the blood mainly red blood cells. The explanation for that really is that the therapeutic levels of Ticagrelor that you see are not sufficient to impact on adenosine uptake because it's a very weak inhibitor of the adenosine uptake pathway known as the MT1. The therapeutic levels are just not getting up to a high enough concentration to have a significant impact on that.

Dr Carolyn Lam:                Stefan, you've thought a lot about this. What did you think of the findings?

Stefan James:                    I think it's very interesting. Of course, the pharmacodynamic effects that you can measure by pretty simple means, the level of platelet inhibition, it should be similar in ACS and stable coronary artery disease and I think it's sort of confirming what Rob has been showing in other populations with ACS ... we have been very interested in trying to understand the additional mechanisms of action of Ticagrelor... try to understand the mortality rate without the benefit for Plato, for example. Was it only -- platelet  inhibition or were there other mechanisms? And, there is a specific Ticagrelor related side effect, dyspnea, which we would have been interested in understanding... is this a  mechanism of action? We can't really explain that.  There are other mechanisms and other effects that we have seen can also be explained by adenosine, so I thought it was very interesting and important to understand more about these mechanisms.  

Dr Carolyn Lam:                Yeah.

Stefan James:                    But I would like to ask you, Rob. Do you think this adenosine hypothesis now, is dead, or should we still try to explore this?

Rob Storey:                        Well of course in this study what we didn't look at was the adenosine kinetics in the tissue level which is where we hypothesize the dyspnea may arise from stimulation of C5 is in the lung tissue so we're missing that piece of information. It's still conceivable that very weak levels of ENT-1 inhibition may impact from adenosine levels in the tissue. We're not seeing a strong ENT-1 inhibition sufficient to raise circulating levels or something that we can pick up on this in vitro assay.

                                                I think it still remains an open question. We've got this sort of contradictory information from drugs like cangrelor and other drugs in development like Elinogrel  where we don't see an impact on adenosine but they still may cause dyspnea.  So I think it's a very open question still.

Stefan James:                    Do you think that your paper gives us additional strength to the hypothesis that the mortality benefit for ticagrelor as seen in Plato is explained by the platelet inhibition and the balance between the reduction in ...

Rob Storey:                        Well I think what we see really in all these studies is that Ticagrelor is a fantastically effective PTY12 inhibitor. It gives you the best level of platelet inhibition during maintenance therapy out of all the available PTY12 inhibitors. And clearly having such more reliable PTY12 inhibition than clopidogrel could still be driving a mortality benefit in high risk patients so we can't exclude the adenosine pathway contributing to some of the clinical effects but I think this sways me a little bit more to the position of thinking this is most of the benefits through platelet inhibition.

Dr Carolyn Lam:                Interesting. So you're on the cutting edge of this. What's the next step then?

Rob Storey:                        Clearly we can see that very effective and reliable P2Y12 inhibition is important and leads to clinical benefits and I think we need to implement that wherever we're using P2Y12 inhibitors. We need to take that message and use a more consistent therapy rather among those with associated with variable response which doesn't seem to make sense. I think this stable PCI population, their risk has fallen. And we see that in this study, quite a number of patients report a response to clopidogrel but no stent thrombosis.

                                                That really reflects, I think improvements in stent design and implantation techniques, so the implication is that maybe aspirin alone is enough to prevent stent thrombosis with modern techniques if you get a good result but in the higher risk patients particularly the ACS patients it's likely you need much more reliable platelet inhibition and that's why Ticagrelor really provides this security.

Dr Carolyn Lam:                So, Rob there is one thing you tested two doses and they seemed to be equivalent at least in antiplatelet inhibition, right? So what does this mean? Should we maybe preferentially use the lower dose from now on, is there still room for the higher dose? Could you share some insights there?

Rob Storey:                        Well I think one has to be cautious in not jumping to adopt a dose just on the basis of pharmacodynamic data but clearly what we show is that the 60 mg dose of Ticagrelor offers a very reliable and consistent level of PTY12 inhibition and that's likely to be very effective in preventing stent thrombosis in combination with aspirin. We also show signals that were also shown in the Pegasus study that the 60 mg dose may be better tolerated such as with lower levels of dyspnea.

                                                So, there is the option for off label use of the lower dose of Ticagrelor in those who cannot tolerate the high dose due to dyspnea because certainly they'll have better platelet inhibition down titrating from 90 to 60 and if they were to switch to Clopidogrel. So I think our study offers some comfort in terms of that aspect. The only caveat is that you have to be careful not to use strong CYP3A inducers such as some epilepsy drugs with Ticagrelor cause that can increase the metabolism and we did have one case of high platelet reactivity with strong CYP3A inducers so using a higher dose initially I think is a good idea. The label says 90 mg for 1 year following ACS and the 6 is licensed beyond one year as a down titration predominantly.

                                                Our study certainly gives some comfort that down titrating earlier if a patient can't tolerate the 90 for whatever reason, seems to be a justifiable thing. And the other thing is the European guidelines support the use of Ticagrelor off label in elective PCI and our study certainly gives some comfort that off label use and the low risk elective PCI patients of the 60 mg dose can be justified at least from a pharmacodynamic point of view.

Dr Carolyn Lam:                Well, thank you because that's exactly what our audience is loving to hear. How do these findings translate into the clinical practice - Would you have any other take home messages for the clinicians listening in?

Rob Storey:                        Well I think one thing we looked at also was troponin release which is very common after PCI. We didn't see an impact of PTY12 inhibition high levels on troponin  release and I think that sort of caveat in terms of that's not going to be the best measure in terms of surrogate for efficacy in the PCI population. The other question really is, how much of the platelet inhibition and how much of the adenosine effects of Ticagrelor influence the clinical outcomes and clearly the studies sways towards the platelet inhibition very consistent high level of platelet inhibition explaining most of the benefits.

Carolyn Lam:                      You've been listening to circulation on the run, don't forget to tune in again next week.


Sep 18, 2018

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. This week's journal features two papers that deal with genetic testing in young athletes and for sudden arrhythmic death, and with findings that may surprise you. They really show the complexities of this era of genetic testing and cardiovascular medicine, and in fact are discussed as growing pains in cardiovascular genetics. You must listen to our feature discussion, which is coming right up after these summaries.

                                                The first original paper this week suggests that targeting fibronectin polymerization may be a new therapeutic strategy for treating cardiac fibrosis. Fibronectin polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblast following cardiac injury. In today's paper, first author Dr Valiente-Alandi, corresponding author Dr Blaxall from University of Cincinnati College of Medicine and Heart Institute, and their colleagues hypothesized that interfering with fibronectin polymerization, or its genetic ablation and fibroblasts, would attenuate myocardial fibrosis and improve cardiac function following ischemia reperfusion injury. Using mouse and human cardiac myofibroblasts, authors found that the fibronectin polymerization inhibitor pUR4 attenuated the pathological phenotype exhibited by mouse and human myofibroblasts by decreasing fibronectin polymerization and collagen deposition into the extracellular matrix as well as by myofibroblast proliferation and migration.

                                                Inhibiting fibronectin matrix deposition by pUR4 treatment or by deleting fibronectin gene expression in cardiac fibroblasts confirmed cardioprotection against ischemia reperfusion-induced injury by attenuating at first left ventricular remodeling and cardiac fibrosis, thus preserving cardiac function. In summary, interfering with fibronectin polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

                                                The Insulin Resistance Intervention after Stroke, or IRIS trial, demonstrated that pioglitazone reduced the risk of both cardiovascular events and diabetes in insulin-resistant patients. However, concern remains that pioglitazone may increase the risk of heart failure in susceptible individuals. To address this, Dr Young from Yale Cardiovascular Research Center and the IRIS investigators performed a secondary analysis of the IRIS trial. They found that older age, atrial fibrillation, hypertension, obesity, edema, high CRP, and smoking were risk factors for heart failure.

                                                Pioglitazone did not increase the risk of incident heart failure, and the effect of pioglitazone did not differ across levels of baseline risk. It should however be noted that in the IRIS trial, the study drug dose could be reduced for symptoms of edema or excessive weight gain, which occurred more often in the pioglitazone arm. Overall, pioglitazone reduced the composite outcome of stroke, MI, or hospitalized heart failure in the IRIS trial.

                                                The next study highlights the importance of genetic variation in cardiac fibrosis and suggests that while fibroblast activation is a response that parallels the extent of scar formation, proliferation may not necessarily correlate with levels of fibrosis. In this paper from co-first authors Dr Park and Ranjbarvaziri, corresponding author Dr Ardehali, from David Geffen School of Medicine, University of California, Los Angeles, the authors utilized a novel multiple-strain approach known as the Hybrid Mouse Diversity Panel to characterize the contributions of cardiac fibroblasts to the formation of isoproterenol-induced cardiac fibrosis in three strains of mice.

                                                They found that isolated cardiac fibroblasts treated with isoproterenol exhibited strain-specific increases in the levels of activation, but showed comparable levels of proliferation. Similar results were found in vivo with fibroblast activation but not proliferation correlating with the differential levels of cardiac fibrosis after isoproterenol treatment. RNA sequencing revealed that cardiac fibroblasts from each strain exhibited unique gene expression changes in response to isoproterenol.

                                                The authors further identified LTBP2 as a commonly upregulated gene after isoproterenol treatment. Expression of LTBP2 was elevated and specifically localized in the fibrotic regions of the myocardium after injury in mice and in human heart failure, suggesting that it may be a potential therapeutic target. That brings us to the end of our summaries. Now for our feature discussion.

                                                We all know that t-wave inversion is common in patients with cardiomyopathy, however up to a quarter of athletes of African descent, and five percent of white athletes also have t-wave inversion on ECG, but with unclear clinical significance despite comprehensive clinical evaluation. Now, what is the role in diagnostic use of genetic testing beyond clinical evaluation when we investigate these athletes with t-wave inversion? Well we're about to get some answers in today's feature paper, and I'm so pleased to have the corresponding author of the paper, Dr Sanjay Sharma from St. George's University of London, as well as our associate editor Dr Mark Link from UT Southwestern.

                                                Sanjay, please let us know what you did and what you found.

Dr Sanjay Sharma:            Well as you rightly say, that up to 25% of black athletes have t-wave inversion, as do three to five percent of white athletes. And these t-wave inversions often overlap with the sort of patterns that you see in patients with hypertrophic cardiomyopathy and arrhythmogenic cardiomyopathy. For example, 80% of people with hypertrophic cardiomyopathy have t-wave inversion as do 60% of patients with ARVC. Now we know that some ECG patterns, t-wave inversions in V1 to V4 are benign in black patients, but the significance of other ECG patterns is unknown. Cascade screening in family members with cardiomyopathy have shown that t-wave inversion may be the only manifestation of gene inheritance, and there are reports to suggest that some athletes with t-wave inversion do go on to develop overt cardiomyopathy. Now when we investigate the vast majority of our patients with t-wave inversion, these are our athlete patients, we don't actually find anything. But over the past decade, also, these has been major advance in next generation sequencing that allows us to perform genetic testing in a large number of genes that can cause diseases, capable of causing sudden death.

                                                And so, we thought we'd investigate the role of this gene testing in athletes with t-wave inversion. We looked at a hundred, 50 black athletes and 50 white athletes who had t-wave inversion, and we investigated them comprehensively with clinical tests. But we also added in a gene panel looking at 311 genes implicated in six cardiac diseases, notably hypertrophic cardiac myopathy, arrhythmogenic cardiomyopathy, dilated cardiomyopathy, left ventricular non-compaction, long QT syndrome, and the brugada syndrome. We found that 21% of our athletes were then diagnosed with a cardiac disorder capable of causing sudden death, and the vast majority of these people had hypertrophic cardiomyopathy. And this diagnosis was based on clinical evaluation. When we looked at gene testing, we found that gene testing only picked up a problem in 10%. So, the diagnostic yield of gene testing was half that of comprehensive clinical investigation.

                                                When we actually looked at athletes who had nothing wrong with them in clinical investigation, and actually had a gene mutation, we found that only 2.5% of athletes who had t-wave inversion but clinically normal tests, actually had something wrong with them. And our conclusions were that gene testing picks up only half the athletes that clinical testing does, and gene testing is only responsible for identifying 2.5% of athletes with t-wave inversion, where clinical tests are negative. That was the summary of our study in short. We did find that black athletes were less likely to have a positive diagnosis of cardiac myopathy than white athletes, and black athletes are also less likely to have a genetic mutation capable of causing a cardiomyopathy than white athletes.

Dr Carolyn Lam:                First and foremost, congratulations on such a beautiful paper, and so wonderfully summarized as well. It really seems to fly in the face, doesn't it? Of the way we've been discussing personalized medicine and saying that we're going to start whole genome sequencing everyone and that's going to provide all the answers for future disease risks. I mean, if I'm not wrong, what your paper is trying to tell us is that at this moment we don't have good examples where genetic testing may trump clinical diagnoses, and in fact we should be still focusing on a comprehensive clinical evaluation of patients and in the absence of a genotype we should learn to question what we're doing in genetic testing. Do you agree with that?

Dr Sanjay Sharma:            You couldn't have said that more precisely. As I've said, the diagnostic yield of clinical testing was 21% versus only 10% with genetic testing. The diagnostic yield of pure genetic testing in people with otherwise completely normal findings clinically was only 2.5%. And the other thing that I forgot to tell you was that genetic testing, if we included genetic testing in addition to comprehensive assessment, cost us three times as much as clinical investigation on its own, and had we relied solely on genetics, and nothing else, it would have cost us ten times more than clinical testing. So our cost per making a diagnosis using genetics only would have amounted to $30,000 per condition.

Dr Carolyn Lam:                Wow, what a great wake up call. Mark, you've thought a lot about this and in fact there was another paper in this week’s journal that has very complimentary messages. In fact you invited an editorial by Dan Roden, and I really loved his title of it, "Growing Pains in Cardiovascular Genetics." Would you maybe add your thoughts in relation to the other paper, as well as overall?

Dr Mark Link:                     Sure. Circulation was very interested in these papers. These are really  ... Now, as Dan Roden says, "Growing pains." Twenty years ago when genetics came out it was looked upon as it was going to completely change our clinical medicine and precision medicine is really relying a lot on genetics. And while ultimately that may be the case, we are in a stage now where the honeymoon is over. And the other paper that was in this same issue was a paper by Hosseini  and colleagues, and it was the Clin Gen paper looking at the Brugada Syndrome abnormalities. Now the Clin Gen is an NIH sponsored group that takes individuals from a number of different institutions and actually gene testing, and tries to provide an independent assessment of the abnormality of genes. Previously is was companies that did this. A company would gene test ... They would look for gene abnormalities, try to link it with clinical disease, and they could basically then do just on their patients. But Clin Gen now is trying to tie all those companies together to get a broad consortion and to look at genetic abnormalities and whether they're truly pathologic, where there's areas of unknown significance, or whether they're truly not pathologic.

                                                So as an example, they took Brugada Syndrome, and they took the different gene abnormalities that have been described from basically different companies and different labs and different institutions, and they looked at the evidence behind the fact that they were truly pathologic, 'cause all 21 genes were defined as pathologic. They found in their independent assessment that only one ended up to be truly pathologic, and the others ones were disputed. And sort of another wakeup call that just because a single company calls a gene pathologic or Brugada Syndrome, does not make it pathologic necessarily. So we all thought these were two very important papers that looked at some of the limitations of genetic testing. We asked Dan Roden, who is really a very accomplished scholar in this field, to provide perspective on this. And I agree, I loved his title, "Growing Pains in Cardiovascular Genetics." And what he did is reviewed the history of genetic testing, and he actually starts before genetic testing and starts with Mendelian genetics, and [inaudible] genetics. And then 23 years ago they started linking that Mendelian genetics to gene abnormalities, especially in diseases such as long QT syndrome and hypertrophic cardiomyopathy.

                                                We've come a tremendous way in diagnosing gene abnormalities and associating them with these underlying cardiac myopathies and hind channel abnormalities. So no one doubts we've come a tremendous way, but there's a long way to go in terms of getting better diagnostic accuracy and really defining where these genetic testing are ultimately going to play out in clinical medicine. So everyone's excited about it, but I think these two papers are two cautionary tales that we do have to remember that genetic testing in 2018 is not the end all and be all.

Dr Carolyn Lam:                I love that, cautionary tales. So important. But where do we go from here? What's the take home message for clinicians listening to this today in 2018? I mean is it that perhaps when we do these things we now need to include medical geneticists and genetic counselors as vital partners as we look at this all? Perhaps we need to not forget the primacy of clinical evaluation. What do you think, Sanjay?

Dr Sanjay Shar:                  Well, there are guidelines from the American Medical Genetics side as to what one defines as a disease-causing mutation. But I agree that we need to be using certified laboratories that can actually interpret the genetic mutations. For example, in our study of athletes, 63% actually had variance of undetermined significance. So they had spinning mistakes in their genes which probably didn't account to anything at all, but had these mutations, or these so called variance of undetermined mutations been interpreted by someone who didn't really know much about this, these could have resulted in false positive results which could cause absolute chaos for an athletes career. So I do think this type of testing has to be governed very, very carefully and needs to be performed in very specialized and certified laboratories.

Dr Carolyn Lam:                Indeed. Not just to the athlete, but to their families too, isn't it? Mark, what do you think is the take home message [inaudible 00:16:18]?

Dr Mark Link:                     I think one of the big take home messages that I took away from these papers is that clinical medicine is not dead. In fact, clinical medicine in this day and age is still the prime way of taking care of patients. Genetic testing is still in its infancy. It doesn't help clinically in too many situations yet. It will in the future. It helps in the diagnosis, it's not as useful in the treatment. So we have a long ways to go with genetics. I like your comment that going forward we're going to need more genetic counselors to make sense of these results. Clinicians are going to have a hard time making sense of these results. I do think that there is plenty of role once a disease causing mutation has been defined, and in that situation it's invaluable in cascade screening in identifying other family members who may be affected, but outside that I do believe and I agree completely with both of you, that clinical medicine is not dead. And clinical evaluation should be number one and should enjoy it's prime time because that's where we still are at. And genetics is still in its infancy and so is cardiology.

Dr Carolyn Lam:                Perhaps in selective settings ... We're not talking here about, for example, hypercholesteremia variance, we're not talking about cancer gene variance for which screening may be a little bit more advanced, and we may understand the gene phenotype associations that are perhaps-

Dr Mark Link:                     I think that understanding gene phenotype associations are going to be critically important in the future. I think, as Sanjay said, the real use of genetic screening now is cascade screening for the family, and there it's invaluable. That you can tell if you've got a co-band with the disease, and with a defined pathological mutation. You can test siblings, sons and daughters, parents to see if any of them have the gene. I think that's where it should be used for sure in 2018.

Dr Carolyn Lam:                Thank you so much Mark and Sanjay. So some precautions, some hope. Very, very balanced discussion. So much more we could discuss, so I really want to highly encourage our audience. Pick up this issue. You have to read these amazing papers and the editorials.

Dr Carolyn Lam:                So, here's a podcast with all your colleagues, and don't forget to tune in next week.


Sep 10, 2018

Dr Carolyn Lam:                We start today's podcast with a few words from our Editor-in-Chief, Dr Joe Hill.

Dr Joe Hill:                           I speak with you today with a heavy heart as we recently lost an esteemed and beloved colleague, Professor Bongani Mayosi. Bongani was a pioneering leader, a renowned investigator, Dean of the Medical School at the University of Cape Town, and an important member of our Circulation editorial leadership team.

                                                Bongani had an abiding passion for the under-served, especially those in his native Africa. He died tragically and suddenly at the early age of 51, just 10 days after recording the podcast you're about to hear.

                                                We mourn the loss of this colleague and our hearts go out to his family. It is a very poignant moment, as we hear his voice once again. We grieve deeply, and are reminded of Bongani's towering achievements and contributions to the betterment of our world.

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore.

                                                CD4-positive T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Today's paper describes the first study to detect apolipoprotein B peptide 18 specific CD4 T cells in mice and humans. First author Dr Kimura, corresponding author Dr Ley from La Jolla Institute of Allergy and Immunology and their colleagues constructed novel P18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotypes by flow cytometry. They found that these P18 specific T cells were mainly anti-inflammatory regulatory T cells in healthy donors, but co-expressed other CD4 lineage transcription factors in patients with sub-clinical cardiovascular disease.

                                                Immunization with P18 reduced atherosclerotic burden in APOE deficient mice and induced antigen specific T regulatory cells. This study therefore, identifies APOB peptide 18 as the first T regulatory APOtope in human atherosclerosis.

                                                The next study suggests that testing intracellular calcium handling in circulating B lymphocytes may be a novel biomarker for monitoring patients with heart failure. During [inaudible 00:02:47] intracellular calcium is released from sarcoplasmic reticulum into the cytoplasm through Type II ryanodine receptor calcium release channels. In heart failure chronically elevated, circulating catecholamine levels cause pathologic remodeling of these Type II receptors, resulting in diastolic sarcoplasmic reticulum calcium leak, thus decreasing myocardial contract [inaudible 00:03:09]. Similarly, skeletal muscle contraction requires sarcoplasmic reticulum calcium release and this occurs through Type I ryanodine receptors. Chronically elevated catecholamine levels in heart failure cause Type I mediated sarcoplasmic reticulum calcium leak, thus contributing to skeletal myopathy and weakness.

                                                In today's paper, first author Dr Kushner. Co-corresponding authors Dr Kitsis from Albert Einstein College of Medicine and Dr Marx from Columbia University, New York hypothesized, that since circulating B lymphocytes express Type I ryanodine receptors, they may be a potential surrogate for defects in intracellular calcium handling due to leaky ryanodine channels in heart failure. Indeed, they found that circulating B lymphocytes from humans and mice with heart failure exhibited remodeled Type I ryanodine receptors and decreased endoplasmic reticulum calcium stores, consistent with chronic intracellular calcium leak. This calcium leak correlated with circulating catecholamine levels. The intracellular calcium leak was significantly reduced in mice treated with S107, which is a drug that specifically reduces ryanodine receptor calcium leak.

                                                Furthermore, heart failure patients treated with LVADs exhibited a heterogenous response. Thus, Type I ryanodine receptor mediated calcium leak in B lymphocytes assessed using flow cytometry may provide a surrogate measure of intracellular calcium handling and systemic sympathetic burden and therefore represent a novel biomarker strategy for monitoring the responses in heart failure therapy.

                                                Hypouricemia and gout are known to be associated with increased risk of cardiovascular disease. And xanthine oxidize inhibitors such as allopurinol and febuxostat are the mainstay of urate lowering treatment of gout, but do they have different effects on cardiovascular risk? First author, Dr Jong, corresponding author, Dr Min from Brigham and Women's Hospital Harvard Medical School in Boston, Massachusetts, studied a cohort of almost 100,000 older Medicare patients with gout and found that there was, overall, no difference in the risk of MI, stroke, new onset heart failure, coronary revascularization are all cause mortality between patients initiating febuxostat compared to those initiating allopurinol. However, there did seem to be a trend toward an increased opiate not statistically significant risk for all-cause mortality in patients who use febuxostat for over three years compared to allopurinol use for over three years. The risk of heart failure exasperation was slightly lower in febuxostat initiators.

                                                The final original paper this week provides important contemporary data on the clinical characteristics in hospital management and long-term outcomes of patients with acute myocarditis. Co-corresponding authors, Dr Ammirati and Kamichi, both from Milan, Italy and their colleagues screened 684 patients with suspected acute myocarditis and recent onset of symptoms within 30 days between May 2001 and February 2017 and included 443 patients with acute myocarditis diagnosed either by endomyocardial biopsy or by increased troponin and edema and late gadolinium enhancement on cardiac magnetic resonance imaging. They showed that among these 443 patients, 118 patients or 26.6% had either left ventricular ejection fraction less than 50% sustained ventricular arrhythmias or a low cardiac output syndrome. While, the 73.4% had no such complications.

                                                Cardiac mortality and heart transplantation at five years was 4.1%, but went up to 14.7% in the patients with complicated presentation and contrast down to zero percent in the uncomplicated cases. Similarly, major acute myocarditis related cardiac events after the acute phase, such as post discharge death and transplantation, sustained ventricular arrhythmias, symptomatic heart failure needing device implantation all occurred in 2.8% at five years, but was much higher in patients with a complicated presentations at 10.8% versus zero percent in the uncomplicated presentations. Thus, the authors concluded that patients with acute myocarditis can be effectively stratified based on their initial clinical presentation. Patients with left ventricular ejection fraction less than 50% at the first echo. Those with sustained ventricular arrhythmias or those with low cardiac output syndrome are at higher risk of cardiac events compared to those without these manifestations.

                                                And that brings us to the end of our summaries. Now, for our feature discussion.

                                                With advances in therapy most deaths in people with HIV are now due to noncommunicable diseases, especially cardiovascular disease. What does the global burden of HIV associated cardiovascular disease really look like? Well we're going to get some answers in today's feature paper. I have with us today the first and corresponding author of the paper, Dr Anubshaw from University of Edinburgh, as well as our associate editor, Dr Bongani Mayosi from University of Cape Town in South Africa.

Dr Carolyn Lam:                Welcome to you both. And Anub, what an important question to examine. Could you tell us how you looked into this question and what you found?

Dr Anubshaw:                   Sure. So, this is a very interesting question from our end and we had in short idea looking at the risk of cardiovascular disease in patients with HIV. And there are many studies of it, varying results. I'm looking at the risk of heart disease and stroke in patients with HIV. So, what we did was a big systematic review to extract all the data out there looking at the risk of heart disease in patients with HIV, we then developed a model that looked at what the overall risk was and then tried to calculate the actual burden of cardiovascular disease attributable to patients with HIV. In some of the work we found, well, primarily we found that the majority of the burden, as expected in Sub-Saharan Africa and that is primarily the cause, in prevalence of HIV is the highest in Sub-Saharan Africa, accounting for about two thirds of all people living with HIV.

Dr Anubshaw:                   The risk of cardiovascular disease with patients with HIV is twofold higher compared to patients not infected by virus. And there was not [inaudible 00:10:12] variations in the actual burden. The majority of the burden in Sub-Saharan Africa and Southeast Asia.

Dr Carolyn Lam:                Wow, Sub-Saharan Africa and Asia Pacific, isn't it? Oh my goodness, Bongani, your views please on these standing results from Africa.

Dr Bongani Mayosi:         Yes. I think these results are actually very important in the Sub-Saharan African region, reaching the, at the center of the HIV/AIDs epidemic in the world. And particularly important now that we are finding people and are on treatment and that they are growing older and there's a thriving proportion of people above the age of 60, they are on HIV infection and therefore the whole question of cardiovascular disease in these patients has become very important and clearly now these data suggest that HIV [inaudible 00:11:08] for cardiovascular disease, but what is more important [inaudible 00:11:14] they are important [inaudible 00:11:17] for cardiovascular disease, but also a [inaudible 00:11:22]. [inaudible 00:11:23] such as another vascular condition, which is pulmonary hypertension associated with HIV detection. [inaudible 00:11:35] with the increase of the number of people on treatment, these particular conditions are becoming [inaudible 00:11:43] in the context of how to [inaudible 00:11:48], but is an important condition in the African continent. So that the overall burden of cardiovascular disease is likely to be greater than is estimated here because the study is only estimating atherosclerotic cardiovascular disease.

Dr Anubshaw:                   That brings up a very intriguing question, Anub. Could you at all distinguish between atherosclerotic risk factors and the role that played versus more HIV specific risk factors, such as the medication, the degree of HIV control, level of inflammation, for example? Now, of course in a meta-analysis this may be difficult, but just your thought.

                                                You're absolutely right from a meta-analysis point of view it's very difficult for a couple of reasons. Firstly, we do not have individual patient level data, so we couldn't really see a [inaudible 00:12:45] level which patients are on [inaudible 00:12:47] therapy and what their personalized risk factors are. Varying schools of thought estimated around the candidates that they need, which kind of portrays a risk of heart disease in the [inaudible 00:12:59] artery in patients with HIV. And what we think may be happening there, one that HIV represents a degree of sub-clinical inflammation that leads to vascular inflammation, which then leads to accelerated atherosclerosis and there's some fantastic mechanistic evidence looking at this where, workers have looked at vascular inflammation in the arteries in patient HIV can go through control and you do get much more vascular inflammation. There is some evidence about the fact that the [inaudible 00:13:31] therapy itself can cause [inaudible 00:13:34] and therefore increase the risk of atherosclerotic heart disease.

                                                And finally, some risky behavior is probably much more, have a look at HIV for example, smoking entered the [inaudible 00:13:46] etc., etc. and there may be a degree of overlap in terms of or correlation in terms of risk factors being much more common in HIV patients, which are more conditional for atherosclerotic heart disease. I think a combination of all those three things probably explain the increase risk of atherosclerotic heart disease and strokes in these patients.

Dr Carolyn Lam:                Indeed. Your paper is so important to raise awareness of that very risk. I mean, if I could please re-iterate, you show very clearly that people with HIV are the two fold increase risks of cardiovascular disease and that that global burden had tripled over the last two decades. I think that your paper really shines a bright light in this area, that we have to study further because the clinical implications are enormous aren't they? Because we're using guidelines developed in non-HIV patients to perhaps treat these cardiovascular diseases in HIV patients and there may be other pathophysiologic mechanisms like you just mentioned. What do you think are the main clinical implications of your paper?

Dr Anubshaw:                   The clinical implication is quite important because what the burden estimate show is that the majority of burden is in no or little information and therefore the resource of those innovations are quite limited, but there's one condition that has been treated so well in these countries. One of the main success stories of medicine, over the last two or three decades and how they've tackled HIV, who runs PEP for has made intrical virals available so widely in the Sub-Saharan African regions, while there's other highly prevalent regions. And they set up logistically clinics to deliver and scare for persons with HIV and if you and I will see that the survival in these patients [inaudible 00:15:39] just mentioned. Then, these patients are at more high risk of other among AIDs related conditions, such as strokes and heart disease. What you now have in these poor resource countries or limited resource countries, where clinics and the logistical support is only set up to deliver cardiovascular risk prevention strategies and therapy. Which is not expensive in terms of antihypertensives, in terms of [inaudible 00:16:06] and in terms of lifestyle factors.

                                                So, I think there is [inaudible 00:16:10] here that the region has to further reduce the cardiovascular burden in this population.

Dr Carolyn Lam:                Bongani, you too recognize the very important clinical implications and in fact invited the editorial by Priscilla Sue and David Waters from San Francisco General Hospital. I love the title of it. Is it time to recognize HIV as a major cardiovascular risk factor? Bongani, what are your thoughts?

Dr Bongani Mayosi:         I think it is time we should be considering the HIV as a risk factor for cardiovascular disease. You know these data arriving from this [inaudible 00:16:48] are quite compelling and when you look, for example at that this is a hot study [inaudible 00:16:55] in the editorial and conferred by HIV, it is almost the same as the other [inaudible 00:17:02]. I mean if you go into it now that in fact the European Society of Cardiology it is already [inaudible 00:17:12] in HIV infected individuals with [inaudible 00:17:19]. So, if now may be entering their [inaudible 00:17:27] of practice, they consider HIV as a significant risk factor for cardiovascular disease and maybe contribute to bring a drug that will modify outcome. I do think though that because of the mechanism of cardiovascular disease it [inaudible 00:17:45] HIV it is not common on the basis of atherosclerotic disease. In Africa as an example, we know very well that the patient tend to [inaudible 00:17:55] with not a lot of traditional risk factors of cardiovascular disease, in fact, atherosclerotic diseases such as [inaudible 00:18:07] still have a relatively low level of [inaudible 00:18:10].

                                                So, we still, I think need to discover what are the other [inaudible 00:18:14] mechanisms that are involved, I mean they do that very much more targeted drug [inaudible 00:18:21] where it needs to be tested, that don't know our traditional interventions for reducing risk and preventing cardiovascular disease. So, there is need for further research here and the mechanisms and specific intervention. That is the important in this large HIV infected populations because at the moment there at least 27 million people in the world, living with HIV who already facing a major public health issue on a global scale.

Dr Carolyn Lam:                Exactly and all these new research efforts, paying attention to this, making sure that we don't underestimate cardiovascular risk and HIV based on traditional risk calculators. All of this starts with awareness and with important papers such as yours, Anub. Thank you so much for publishing that with us at Circulation.

                                                Well, listeners you know how important this is globally, so please share this podcast with your colleagues and don't forget to tune in next week.


Sep 4, 2018

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore.

                                                Current guidelines recommend measurement of one of the cardiac specific isoforms of cardiac troponin complex. However, what's the utility of combining measurements of troponins I and T in the early diagnosis of acute myocardial infarction? Well, you have to wait for our upcoming feature discussion, but it's coming right up after these summaries.

                                                The first original paper this week sheds light on the genetic basis and mechanisms of bicuspid aortic valve, the most common congenital heart defect in the population. We know that bicuspid aortic valve is an autosomal dominant trait with variable expression and incomplete penetrants suggestive of genetic and environmental modifiers. In the current study, first author Dr Gharibeh, corresponding author Dr Nemer from University of Ottawa, and authors of the Bicuspid Aortic Valve Consortium assessed cardiac structure and function in mice, lacking a GATA6 allele. They found that GATA6 heterozygous mice had a highly penetrant type of bicuspid aortic valve with right and left leaflet fusion, which is the most frequent type found in humans. GATA6 transcript levels were lower in human bicuspid aortic valve as compared to normal tricuspid valves. Mechanistically, GATA6 haploinsufficiency disrupted valve remodeling and extracellular matrix composition through dysregulation of the importance in the molecules including matrix metalloproteinase nine. Cell-specific inactivation of GATA6 reveal that an essential rule for GATA6 in secondary heart field myocytes. Thus, the study identifies a new cellular and molecular mechanism underlying bicuspid aortic valve.

                                                In the field of cardiac regeneration, c-Kit positive adult progenitor cells were initially reported to produce new cardiomyocytes in the heart. However, more recent genetic evidence suggests that such events are exceedingly rare. Today's paper provides insights into this discrepancy and it is from first author Dr Maliken, corresponding author, Dr Molkentin from Howard Hughes Medical Institute Cincinnati Children's Hospital Medical Center. The authors took a novel approach of deleting the necessary cardiogenic transcription factors, GATA4 and GATA6, from c-Kit expressing cardiac progenitor cells to determine whether true de novo cardiomyocyte formation would occur. They found that deletion of the necessary cardiogenic transcription factors, GATA4 and GATA6, from these c-Kit+ cardiac progenitor cells remarkably resulted in greater apparent cardiomyocyte derivation from the c-Kit+ cells. Deletion of GATA4 from c-Kit–derived endothelial progenitors altered the integrity of the endothelial cell network in the heart, resulting in greater c-Kit+–derived leukocytes entering the heart and fusing with cardiomyocytes.

                                                Thus, they demonstrated a new role for GATA4 in endothelial differentiation, specifically showing for the first time that GATA4 is essential for vascular development by the c-Kit lineage. The study shows that leukocyte to cardiomyocyte fusion is the primary basis for path lineage tracing results, incorrectly suggesting that c-Kit+ cardiac progenitor cells generated de novo cardiomyocytes in the heart.

                                                Lecithin–cholesterol acyltransferase, or LCAT, is the sole enzyme that esterifies cholesterol in the plasma. Its role in the supposed protection from atherogenesis remains unclear, because mutations in LCAT can cause more or less carotid atherosclerosis. Addressing this conundrum, co-first authors Drs. Oldoni and Baldassarre, co-corresponding authors Dr Kuivenhoven from University Medical Center Groningen, Dr Holleboom from Academic Medical Center Amsterdam, and Dr Calabresi from University of Milano in Italy hypothesized that genetic mutations causing complete LCAT deficiency versus partial LCAT deficiency would be differentially associated with carotid atherosclerosis in carriers of LCAT mutations. To study this, they looked at 74 heterozygotes for LCAT mutations who are recruited from Italy and the Netherlands and who were assigned to complete versus partial LCAT deficiency. These were also compared to 280 controls. Using carotid intima-media thickness as a measure of atherosclerosis, the authors demonstrated that carriers of LCAT mutations leading to complete LCAT deficiency exhibited less carotid atherosclerosis, indicating a reduced risk of cardiovascular disease.

                                                By contrast, however, carriers of LCAT mutations leading to partial LCAT deficiency showed marginally more atherosclerosis. The association of mutations in LCAT with subclinical atherosclerosis appeared to be related to the capacity of LCAT to esterify cholesterol on apoB-containing lipoproteins since the abnormal LCAT present in the partial deficiency was only active on this class of lipoproteins. These important findings bear relevance for pharmaceutical strategists that target LCAT.

                                                After a bioprosthesis aortic valve replacement, what is the incidence, correlates, and outcomes of hemodynamic valve deterioration? First author Dr Salaun, corresponding author Dr Pibarot from Quebec Heart and Lung Institute and their colleagues studied 1,387 patients who underwent bioprosthetic aortic valve replacement and found that hemodynamic valve deterioration identified by Doppler echocardiography occurred in one-third of patients and was associated with a 2.2-fold higher adjusted mortality. Diabetes and renal insufficiency were associated with early hemodynamic valve deterioration whereas female sex warfarin use and stented bioprosthetic valve versus the stentless ones were associated with late hemodynamic valve deterioration. These findings suggest that following bioprosthetic valve replacement, a systematic echocardiographic follow-up may be considered to ensure adequate detection and quantitation of hemodynamic valve deterioration.

                                                That wraps up on the summaries this week. Now for our feature discussion.

                                                We are recognizing the critical role that cardiac troponins play for the early diagnosis of acute myocardial infarction. We also know that there are different isoforms of cardiac troponins, the cardiac troponins T and I. Now, have you ever considered combining the two? How does that help the early diagnosis of acute myocardial infarction? Well, I am delighted to have with us the corresponding author of our feature paper today, Dr Christian Mueller from University Hospital Basel in Switzerland, a very familiar voice on this podcast. Welcome, Christian, and thank you so much for publishing yet another wonderful paper with us.

Dr Christian Mueller:      Thank you very much for highlighting this important work and allowing me to comment on it in the podcast.

Dr Carolyn Lam:                Christian, first of all, could you paint the background to help us understand what's the difference between the two isoforms, I mean, in terms of diurnal variation, the way that they may be released earlier or later, the way they may or may not be impacted by comorbidities like renal dysfunction or hemolysis? Could you help us understand why there may be rational to combine the two in looking at their impact on the diagnosis of acute myocardial infarction?

Dr Christian Mueller:      The measurement of cardiac troponin as a structural protein unique to the heart clear is a central piece in our early diagnosis of acute myocardial infarction, so both for the early rule out in patients who present with chest pain and are finally found to have more benign disease as well as the early ruling. In general, I think it's important to highlight that there are two isoforms exactly as you have mentioned, so there is cardiac troponin T and cardiac troponin I. So these two proteins are cardiac specific and are used in the diagnosis of acute myocardial infarction. Now with the development of high-sensitivity methods or measurements of both cardiac troponin T and cardiac troponin I concentrations, we have been able to get a little bit of a better understanding of in fact differences in the pathophysiology as well as analytical details between cardiac troponin T and I.

                                                Before I start highlighting the differences, I think it's important, I mean, both signals show a very strong correlation, so still very, very similar to each other. However, the small differences that have begun to emerge kind of allow to suggest that possible we could use them together as two pieces of information in the diagnosis.

                                                So, what are the differences? First, exactly as you have highlighted, that if in fact that diurnal rhythm with cardiac troponin T, which means that cardiac troponin T concentrations are higher in the morning hours as compared to the evening, we still have no clue why that's the case, but it's a relevant difference about 25% and it has been shown in two cohorts and a group from Maastricht who was the first one highlighting this. This rhythm has not been found for cardiac troponin I. The second difference is that, again, probably understood in many, many population studies cardiac troponin T concentrations are even stronger predictors of death as compared to cardiac troponin I concentration. Then the third difference it seems that if we measure it with high-sensitivity assays, for example high sensitivity, it seems to rise or if you released from injured cardiomyocytes even slightly earlier as compared to T and possibly even less injuries necessary to release I as compared to T.

                                                Then you mentioned renal function. Cardiac troponin T concentration shows slightly higher correlation with renal function as compared to I. Also, other pre-analytical issues, hemolysis seems to affect T and I concentration in a different way. So a lot of small tiny differences that have emerged and that underlie the hypothesis that possibly by combining the two signals we could be even more accurate in the diagnosis rather than relying on one on its own.

Dr Carolyn Lam:                That's good. That really sets up the rational very well. I think in and of itself is a learning lesson, because I think most clinicians sort of take the two equivalently. So could you tell us what you found?

Dr Christian Mueller:      I would like to of course thank the fantastic team that has allowed us to generate this data. It's a collaboration between the APACE investigators, the ADAPT investigators and experts in clinical chemistry from Maastricht University and Noreen Fandalin and Karen Villa of the first office. So we used two large diagnostic studies, APACE and ADAPT. We measured high-sensitivity cardiac troponin T and I and both of them and compared the diagnostic performance as compared to the final adjudicated diagnosis by two independent cardiologists who, of course, had all information, cardiac imaging and whatever you need to adjudicate.

                                                So, what we found is that in general if you look at diagnostic accuracy, overall is quantified by the area under the curve. Combining the two signals did not consistently increase overall diagnostic accuracy as compared to the individual isoforms. However, we were able to document some improvement for the rule out for the very early rule out of acute myocardial infarction. So the concept that is extremely attractive of course from a medical as well as from an economic perspective is to rule out the presence of acute myocardial infarction with a single blood draw. So, we can do this if we assess the ECG. The ECG doesn't show relevant changes. Then if the troponin concentration measured with a high-sensitivity assay is very low, then the likelihood that the patient would have an acute myocardial infarction again is extremely low or in scientific term sort of a negative predictive value approach is 99 to 100%. By combining very low concentration for high-sensitivity T and very low concentration for I, we were able to increase the efficacy of the early rule out and that seemed to be the most likely possible clinical utility of combining the two signals.

Dr Carolyn Lam:                Even that so-called neutral findings are very important. It's an important question to ask and important answer to get. Could you give us an idea for the rule-out part? How much do we gain? How much exactly do we gain by using both assays instead of just one?

Dr Christian Mueller:      So, the efficacy of the early rule-out depends to some extent on the assay used and the cut off applied. So the current you see algorithm uses cut-off that has been shown to be very safe. However, they are regarding their efficacy not very high. So the current you see recommended cut-offs and approach, allows the rule-out only in about perhaps 10 or 15% of patients. That number can be significantly increased, likely doubled or perhaps even increased threefold by using the combination approach. So this has been consistently showed both in the derivation and the validation cohort.

Dr Carolyn Lam:                Yeah. Do you think this is ready for prime time? I noticed a very balanced discussion actually calling for future studies, but perhaps you could state it better now.

Dr Christian Mueller:      The main limitation regarding prime time is the fact that currently manufacturers either of a high-sensitivity TSA or of a high-sensitivity high method, which means that the vast majority of hospitals at this point in time do only have one method available. It would require quite substantial investment in both hardware as well as changing of the logistics in the lab to implement measurement of both assays. So I think it's likely feasible, but it would be associated with relevant investment from a hospital perspective. In addition, I mean, also the rule-out approaches that use of only one assay also there are studies ongoing in trying to further increase the efficacy of the single marker approach. So I think it's the best tool marker strategy that we were able to come up with recently, because many of the other biomarkers that we had tested really didn't work out. Still, as you mentioned, I think it's also important to be very, very honest that it will be difficult to implement tomorrow in most institutions.

Dr Carolyn Lam:                Yeah, and perhaps a little bit more work needs to be done to sort first identify perhaps special situations where these may be particularly helpful. I supposed like you just said when we're thinking of the ESCs to review one-hour type algorithm, who knows maybe we should be having that extra insurance of the second test in those that test it negative in the first or something like that. Do you plan further work? I always ask you because you're always in the forefront of these things and we just love touching your work.

Dr Christian Mueller:      We have several additional analyses ongoing. Again, I think the main part is for just to go ... I go back from a clinical perspective. So I think for many hospitals that are using T at the moment, it's important to have I available for certain situations. So for example if you have a patient in whom you have evidence of chronic skeletal muscle disease, most of these disorders are rare but some of them have been shown to be associated with increasingly highly troponin T that do not seemed to be related for cardiac diseases but from skeletal muscle. This is rare but if you have a patient with that kind of history, then the dual mark measurement is I think mandatory.

                                                The same applies to iso that the other reasons to have false positive results for iso whenever you are ... If your hospital is using I, you should have the T method also available because once in a while you will identify patients in whom you have an I result that doesn't really match the clinical setting, then it's so easy and often so helpful to get the T result to decide on the most appropriate measurement of patient.

                                                For which patients are kind of a standard that measures T and I would be justified, I think that's something to tease out in future study. I think that the rational is there and likely it will depend also on kind of which T or which I method we might use in the future. So at the moment, we have one method for high-sensitivity T, but there are several other methods in development and kind of applying for FDA approval for high-sensitivity I and possibly combination of these might be even more beneficial regarding the single measurements and I think that has to be teased out in future studies.

Dr Carolyn Lam:                Exactly, but what great insights for us to consider as clinicians now for specific cases where we may consider find those if we have those in our institutions. At the end of the day, I supposed cost-effectiveness analysis will need to be done. Agree?

Dr Christian Mueller:      Absolutely, absolutely. The good thing about troponin, it's extremely inexpensive. So as compared to most of the new fancy biomarkers that are usually, rather prices of troponin is a routine marker. It's inexpensive. It's there for very likely that if we are able to document some clinical value that also the cost-effectiveness study that's definitely unnecessary will show also some economic benefit.

Dr Carolyn Lam:                Oh, Christian, thank you for publishing yet another impactful and clinically relevant paper with us here in Circulation. I mean, it's exactly the kinds of papers that we really treasure here, because they directly inform clinicians and open our eyes to actually things that we should be considering in our everyday practice. Clod I ask you maybe cheekily to share about your experience with publishing at Circulation? Someone like you will be the best person to tell the world what it's like.

Dr Christian Mueller:      Oh, of course. I mean, for us as a research group and for me as a researcher, it's fantastic. It's perfect to have some of our work published in Circulation that has fantastic impact factor, fantastic readership and ensures that the research catch the attention that's fantastic. Also, I think for us as a research group, the recognition of being able to publish in Circulation is outstanding and it helps us continue in the research group that we do. The comments made to large extent also by the editors. Also, on this manuscript, I think we're incredibly insightful and definitely had a major contribution to the final product to make it as attractive and also as balanced and insightful I think as it is at this point in time.

Dr Carolyn Lam:                Thank you so much for providing that feedback, because it is our aim, explicit aim to put a partner authors in getting the best of the manuscript and working really closely with you. So thank you once again, Christian, for your time today. Audience, I know you've heard many times from this favorite person that we have on our podcast.

                                                Do share this podcast with all your colleagues and don't forget to tune in again next week.