iPSC-derived cardiomyocytes to model estrogen receptor modulation of stress cardiomyopathy and arrhythmic syndromes

It has long been known that young women are relatively protected from heart disease compared to men. For sudden death arising from disrupted heart rhythm, men are 80% more likely to be affected. This often occurs after sudden or chronic stress, and is triggered by adrenaline. We have been studying a newly described syndrome in which a sudden extreme shock (accident, bereavement) produces not sudden death, but a temporary reduction in heart function which then reverses. Sufferers come in to hospital thinking they are having a heart attack, but recover within days to weeks to have normal heart function. Interestingly, 80-90% of sufferers are women around or after the menopause. We reproduced this in an anesthetised rat model, and showed that a single dose of adrenaline could give the same effect. (In fact one dose of adrenaline from an epi-pen can sometimes have the same result in people). We found that very high adrenaline could switch from being stimulant to depressant, through an effect on its receptors on the heart cell surface. When we tried to stop it being depressant on the heart function, we induced sudden cardiac death. Our idea is that high adrenaline goes from being stimulant (but damaging) to depressant (but protective) through a receptor switch, and that this protects against sudden death. Here we want to know why this happens particularly in women at a time when estrogen is dropping, and whether this can give us insights into male/female differences.

For these experiments we want to move away from animal models to human pluripotent stem cells (hPSC). We know hPSC can reliably be turned into beating cardiac cells (hPSC-CM), and that they can show the same disrupted rhythm as a patient from which they came. They have adrenaline receptors more like human than are found in animal species, and adrenaline can produce the same kind of rhythm effects in the dish as in the whole heart. They are also very long-lasting in culture, and can be more easily experimented on - for example to turn off and on specific proteins. We already have ways to measure their beating activity and how easily they can be damaged. We also have a number of hPSC lines reflecting different natural gene variation in human populations. We aim to discover the mechanism of the adrenaline switch and how it can be changed by female hormones. The ideal goal for us would be to find a way to harness the protective effect without producing the temporary loss of heart function, and to develop a drug to use that mechanism. One other advantage of the hPSC-CM is that they can be produced in large quantities and used in multiple assays at the same time. The same cells and methods are being used now by pharmaceutical companies to test drugs, which means that any of our promising findings can go more quickly into drug development.

Our hypothesis is that beta2AR switching from Gs-adenylyl cyclase-cAMP signaling to Gi-p38 MAPK signaling decreases cardiac contraction at the same time as conferring protection against arrhythmias and cell damage, and that estrogen signalling modifies this. The aim is to investigate the effect of estrogen on the biased agonism of the beta2AR and the relationship of this to the genotype of the SCM patient. We will investigate separately the cardioprotective (anti-arrhythmic, cell survival) and cardiodepressive effects of the beta2AR -Gi switch using human pluripotent stem cell derived cardiomyocytes (hPSC-CM) from human embryonic or induced pluripotent stem cells.

Contraction, calcium transient and action potential duration will be used as readouts of function and to detect arrhythmia occurrence (early or delayed after-depolarisations). cAMP levels will be measured using transfected EPAC2 FRET sensor. These methods have been validated, are in routine use in the UK laboratory, and will be scaled up to high throughput assays in most cases. Cardiotoxicity experiments for apoptosis and necrosis will use the high content automated microscopy system, Cellomics ArrayScan, with readouts including loss of mitochondrial membrane potential, caspase 3 activation, nuclear condensation, increased cell permeability and decreased cell number. We are developing in a parallel project a number of hPSC-CM lines, either by CRISPR gene editing or by reprogramming of lymphoblasts from available subjects with the correct haplotype. These include lines overexpressing the main beta2AR SNPs with fluorescent (SNAP tag) labelling to allow monitoring of beta2AR movement during catecholamine/beta-blocker challenge.

Our goal is to find a difference in signalling between cardiodepression and cardioprotection which could be exploited in design of beta2AR -Gi agonists to produce an antiarrhythmic and cardioprotective effect without depression of cardiac function.

The main potential impact would be in terms of drug development. As stated in the application, the ideal outcome would be to find a difference in signalling between cardiodepression and cardioprotection which could be exploited in design of beta2-Gi agonists to produce an antiarrhythmic and cardioprotective effect without depression of cardiac function. Sudden cardiac death involving catecholamines is an immense clinical problem, occurring in babies and young adults with genetic syndromes, contributing to mortality during myocardial infarction and accounting for around half of the deaths in chronic heart failure. Anti-arrhythmic drug therapy has been notoriously hard to develop: the mainstay is beta-blocker agents, but even these are either not tolerated or ineffective in a number of patients and problematic to use prophylactically in younger subjects. Implantable cardiodefibrillators are also effective, but carry a burden of invasive intervention and inappropriate (and distressing) shocks. The incidence of sudden death in genetic syndromes is most frequent around and after puberty, implying a hormonal component. The protective effect of estrogen is well recognised, but hormone replacement therapy has been complex even in post-menopausal females and is unacceptable for males. To understand the beneficial effect of estrogen in terms if its modulation of beta2AR signalling could be a new avenue for drug design.

Our work will also have implications for clinicians in understanding the Stress Cardiomyopathy syndrome and the complex pharmacology of this, since our hypothesis would not only exclude catecholamines and phosphodiesterase inhibitors as treatments but some beta-blockers which we have shown to biased beta2-Gi agonists (Paur, Circulation 2012). While the syndrome is largely benign in outcome there are complications in a proportion of cases, and there is certainly the possibility of exacerbating the cardiodepression by choosing the incorrect treatment.

One highly desirable outcome for the field and beyond would be for the blanket use of adrenaline in a number of conditions, such as refractory asthma, trauma, sepsis and resuscitation, to be reconsidered. It is often found that patients become "inotrope-unresponsive" to heroic doses of adrenaline, and we suggest that this is a result of the beta2 Gs to Gi switch now producing negative inotropic effects through adrenaline. Adrenaline at this point is doing more harm than good in terms of supporting cardiac function, and might be better replaced with a non-cAMP-related inotrope.