The Cell Electrophysiology Lab
The Cell Electrophysiology Lab is based at the Imperial College London National Heart and Lung Institute, Myocardial Function and is part of the British Heart Foundation Cardiovascular Regenerative Centre. Research within the laboratory aims to investigate the electrophysiology of heart failure and the consequences of treatment on the contractile and electrical properties of the heart. One major interest of the lab is the process of calcium regulation which links the electrical and mechanical activity of the cardiac myocyte (excitation-contraction coupling).
The group’s research focuses on the mechanisms of myocardial regeneration following mechanical device or stem and gene therapy, and the cellular and molecular mechanisms of arrhythmias. The major themes are the effects of changes in mechanical load on myocardial structure and function, the role of heterocellularity in cardiomyocyte physiology and disease, the plasticity of human pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) in response to biological and physical stimuli, iPSC-CM integration in adult myocardium and the study of suitable cardiac tissue engineered constructs, the identification of factors that control structure and function in myocardial tissue. These aspects are studied using different models: single cells such as isolated cardiomyocytes, combination of different cell types (cardiomyocytes and fibroblasts or endothelial cells), combination of cells with three dimensional scaffolds, multicellular preparation such as myocardial slices, and Langendorff-perfused and normally loaded working whole heart and in vivo animal models.
Selected publications from the Cell Electrophysiology Group in the last few months:
- Watson SA, et al. - Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro - Nature Communications 2019
- Kane C & Terracciano CM Human Cardiac Fibroblasts Engage the Sarcoplasmic Reticulum in Induced Pluripotent Stem Cell-Derived Cardiomyocyte Excitation-Contraction Coupling. Journal of the American College of Cardiology 2018
- Watson SA, et al. - A reproducible method for the preparation of highly viable adult ventricular myocardial slices for acute & chronic studies – Nature Protocols. 2018
- Perbellini,F et al.- Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue – Scientific Reports. 2017
- Perbellini F, et al.- Investigation of cardiac fibroblasts using myocardial slices – Cardiovascular Research. 2018
- Kane C & Terracciano CM Chamber specificity Stem Cells 2017
- Mawad D, et al. - A conducting polymer with enhanced electronic stability applied in cardiac models – Science Advances. 2016
Heart failure is a common, costly and potentially fatal condition. In 2015, about 40 million people were affected globally, with numbers rising steadily due to increased survival from acute diseases and aging. Heart failure occurs when the heart is no longer able to pump enough blood to meet body requirements. This results in fatigue, breathlessness and a gradual deterioration of other organs such as kidneys, liver and lungs. 50% of patients with heart failure die of irregularities of heart beat (arrhythmias), making this condition both an electrical and mechanical problem. The Cellular Electrophysiology Laboratory, run by Professor Cesare M Terracciano, has been investigating heart function and particularly heart failure for more than 15 years.
Why is it so difficult to cure chronic heart failure? In the vast majority of cases heart failure is progressive and incurable. Using novel drugs and surgery it is now possible to manage some of the symptoms and complications of the disease but the underlying damage of heart tissue responsible for the disease cannot be repaired. Following a heart attack, which kills several millions of muscle cells (myocytes), the body is unable to replace the dead cells, even if the cause of the heart attack (blockage of a heart vessel) has been removed. Similarly, cardiac cell death can result from familiar diseases, chronic high blood pressure, diseases of the heart valves, congenital defects, whilst in certain patients the exact cause is unknown. Following cell death, a scar is formed which does not contribute to blood pumping. The surviving cardiac myocytes respond to the increased workload by augmenting their individual size (hypertrophy) and function in a process of exceptional plasticity and adaptability, which is lifesaving in the short term, but with time brings about damage to these cells with loss of function, further cell death and worsening of the condition. Thus, chronic heart failure is effectively a self-inflicted disease and the consequence of adaptation of the heart to injury. This, not the original injury, is the central problem of heart failure and the major hurdle to finding a cure.
To cure heart failure several strategies have been attempted all of which have had limited success so far. The most obvious, radical and difficult strategy to achieve is the formation or insertion of new cardiac myocytes in the heart (cardiac regeneration). Despite an enormous number of studies in this field, it has not been possible to date to induce a sufficient regeneration of heart muscle (see below). Another possibility is to prevent the chronic damage of the surviving cardiac myocytes so that they can remain in a compensated, stable state. This strategy is hindered by the poor understanding of the processes involved in the plastic response, with several theories (genetic, metabolic, post translational regulation etc.) constantly gaining/losing popularity among scientists. For this reason, a targeted, definitive strategy has not yet been designed, with several studies based on empirical theories showing limited or no benefit in the treatment of heart failure. A pressing need for fundamental studies exists to understand myocardial plasticity and adaptability. Finally, a promising strategy is to reduce chronic overload directly while maintaining adequate circulation of blood. This can be achieved using mechanical pumps, ventricular assist devices (VADs). We and others have own that VADs induce a substantial improvement in heart structure and function, but their exclusive use in terminal heart failure patients as a bridge to transplantation, the high number of complications and high financial cost have limited the evaluation of this strategy for the treatment and cure of heart failure.
The term plasticity is used to describe changes in cell/tissue/organs in terms of morphology, function, appearance and metabolism, as a response to changes in the environment.
Heart tissue shows a very effective and sophisticated adaptability and plasticity that allows for survival in the most disparate environments and situations. The response to changes in mechanical load is one of the most studied and the Cellular Electrophysiology group has been investigating how this can be used for the treatment of heart failure.
VADs are mechanical pumps that unload the left ventricle. In recent years, VADs have been used as a temporary strategy to sustain the circulation of patients in end-stage heart failure until transplantation. Clinical and laboratory studies have shown that these devices induce beneficial changes in heart structure and function, a phenomenon termed reverse remodelling. In some patients, the improvement in function is so significant that the devices can be explanted without the need for transplantation (Bridge-to-Recovery). This concept is revolutionary as it suggests that heart failure is reversible and indicates an alternative to transplantation for patients in end stage heart failure. While the prospect that VAD therapy might be curative has enormous potential, the clinical experience of VAD-induced recovery is sparse. The reduction in biomechanical load induced by VADs has a complex effect on the heart which appears to be time - and aetiology - dependent. During VAD treatment, mechanical unloading may be beneficial, inducing reverse remodelling, but prolonged use may also lead to negative consequences, termed ‘myocardial atrophy’. While clinical studies continue to address the feasibility of VADs as a ‘Bridge-to-Recovery’, there is a pressing need to refine this modality of treatment.
Over the last few years, we have performed several studies aimed at understanding the mechanisms by which mechanical unloading is able to induce reverse remodelling of the failing ventricle. We were among the first to show that myocytes isolated from patients treated with VADs have improved contractility and calcium handling, and that specific electrophysiological changes are associated with clinical recovery (Terracciano et al., Circulation 2004).
Cardiomyocytes adapt to overload or unload with changes in their morphology and function. T-tubules are a critical structure in ventricular cells, as they mediate the interaction between L-type calcium channels and ryanodine receptors (the channels responsible for release of intracellular stores of calcium) and define regulatory microdomains. This interaction underlies the phenomenon of calcium-induced calcium-release. We have shown that mechanical unloading alters cell architecture with important consequences for the electrophysiology of cardiomyocytes (Ibrahim et al., FASEB Journal 2010).
Currently, Dr Sean Bello, a cardiac surgeon with a specialist interest in cardiac transplantation, is performing a BHF funded project to investigate the effect of mechanical unloading on the regenerative capacity of the adult heart. Furthermore, by using reperfusion and prolonged unloading in a rat model of heterotopic abdominal heart transplantation, he is investigating the effect of this combined strategy on the propagation of myocardial fibrosis after a heart attack. His investigation involves a range of techniques to identify changes in collagen amount and localization, cellular remodelling and cellular proliferation. He is also carrying out pressure-volume loop studies to assess ex vivo rat ‘whole heart’ systolic and diastolic functional performances after unloading using a Radnoti working heart apparatus.
Another important aspect of our research is the study of the functional consequences of changes to cellular architecture in disease and after therapeutic interventions. We discovered a previously undescribed role for changes to one of the cytoskeletal proteins, protein 4.1, in modifying the electrophysiology of cells with possible roles in disease (Stagg et al., Circulation Research 2008).
We are now developing a new methodology that can be used to investigate structural and functional responses of cardiac cells to prolonged changes of mechanical load within the multicellular environment in vitro. Using a viable multicellular preparation called myocardial slices (300µm thick sections of cardiac tissue) Samuel A Watson, a MBBS/PhD student, is studying these changes in terms of cellular morphology, contractility, gene and protein expression.
Ifigeneia Bardi, a PhD student funded by the NHLI Foundation, studies the role of mitochondrial function in response to different degrees of mechanical load in the heart. Mitochondria are the energy factory of the cell, determining the amount of energy production in response to the cell’s demands. The function of mitochondria could determine the consequence (beneficial or not) of cardiac load. In our studies, we are using living myocardial slices to evaluate metabolism, mitochondrial structure and function using imaging, biochemical and molecular techniques.
See below for the use of naïve cells, and iPSC-CMs in particular, to study plasticity.
Heterocellularity: cardiac fibroblasts and endothelial cells
While the myocyte is the workhorse of the heart, endothelial cells and cardiac fibroblasts are a crucial component of the myocardial syncytium, respectively forming 60% and 15% of the total cardiac cell number. Traditionally seen as having a supportive role next to the functional activity of cardiomyocytes, fibroblasts produce and regulate the turnover of the extracellular matrix in healthy myocardium and play an important role in cardiac remodelling in disease. This is particularly interesting when seen in the mechanical unloading of the heart using VADs. While there are substantial functional benefits of mechanical unloading, adverse effects include significant myocardial fibrosis. We have recently shown that the pacemaker current inhibitor Ivabradine enhances reverse remodelling in mechanically unloaded rats, improving both function and cardiac fibrosis independently of heart rate (Navaratnarajah et al. Cardiovasc Res 2013). A major interest of the lab now is to identify if Ivabradine is producing these effects through manipulating cardiac fibroblast function, and identifying mechanisms through which this is achieved.
More recently however, appreciation of the direct effects of fibroblast activity on cardiomyocyte function has grown. As key regulators of the extracellular matrix and a potent source of cytokines and growth factors, changes in the activity of these cells have important consequences for heart function in both health and disease. We have recently shown the importance of considering the regulation of cardiomyocyte electrophysiology in a multicellular environment, with rat cardiac fibroblasts from diseased hearts having substantial effects on cardiomyocyte calcium handling properties through TGF-β mediated pathways (Cartledge et al. Cardiovasc Res 2015).
The group, in particular Brian Wang, a MBBS/PhD student, is now investigating the role fibroblasts play in cardiomyocyte excitation-contraction coupling - in particular the modalities by which fibroblasts and cardiomyocytes can interact, namely via secreted factors, direct contact between cells or through modulation of the matrix surrounding the two cell types.
For many years, endothelial cells were thought to be 10% of the total cardiac cellular population and to provide a mere “passive lining” supportive role in the heart. In the past few years, it’s been recognized that endothelial cells form the most abundant cell type in the heart (60%; they outnumber cardiomyocytes 3:1) and are a dynamic regulator of vascular tone and growth/function of nearby cells. Our laboratory is trying to better characterize these effects - in particular Oisín King, a PhD student with a background in Mechanical/Biomedical Engineering and Biomaterial Synthesis, who is interested in the development of biomimetic in vitro tissue models, and the use of these models to better understand cell-cell interactions, particularly the interaction between endothelial cells and cardiomyocytes. Using 3D in vitro tissue models, he is investigating how different methods of cellular interaction modulate cardiac function and contractility.
Jerome Fourre is a PhD student with a background in cell electrophysiology. His work is now focused on the interaction between endothelial cells and cardiomyocytes during inflammation, and investigating the mechanisms behind cardiac damage in vascular diseases. In this context, he is establishing a spectrum of in vitro co-culture models while also studying the effects of cardioprotective signalling pathways and gene regulation on this intercellular cross-talk.
The goal of our research is to increase our understanding of the effects of fibroblast and endothelial cells on cardiomyocyte function, assess pathways through which these effects are achieved and ultimately identify mechanisms that could be manipulated to improve cardiac function in disease.
Regeneration, integration, tissue engineering
Regenerative medicine is a branch of translational research which deals with the process of regenerating/replacing cells, tissue or organs to restore their normal functions. This idea is particularly promising for those organs, such as cardiac tissue, with extremely limited regenerative capacity. Despite the potential and great appeal of this method, the real efficacy of cell therapy is still debated. The cell type used for transplantation, the delivery method and cell retention, and the development of dangerous heart rhythms (arrhythmias) are some of the main obstacles that researchers are trying to overcome. One of the main challenges has been to develop methods to image and understand how injected cells integrate and differentiate in the host tissue. Dr Filippo Perbellini is a Research Associate at Imperial College London and part of the British Heart Foundation Cardiovascular Regenerative Medicine Centre. His research interest is focused on myocardial slices: a multicellular preparation with preserved structural, biochemical and electrophysiological properties. He is using myocardial slices as a platform to study the mechanisms of functional integration and electrical coupling of transplanted cells with the recipient myocardium.
To investigate these processes, he is using several imaging and functional acquisition methods, and he has also developed new tissue clearing imaging methodologies for three-dimensional visualization of cardiac tissue.
Among the different cell types, we are interested in the physiology of human iPSC-CMs. These cells, for their unlimited source, human origin and patient-specificity and lack of ethical concerns, are among the most interesting platforms for heart regeneration. We are using these cells to define plastic responses and cross talk with other cell types. We have analysed their ability to develop a chamber-specific phenotype and defined criteria to assess this (Kane Stem cells, Du Biophys J).
Regenerative medicine also includes the possibility of growing artificial tissues in the laboratory and implanting them when the body cannot heal itself. Eleanor Humphrey, a PhD student, has been working on the observation that iPSC-CMs differ significantly from adult cardiomyocytes in both their structure and function, limiting their use both in vivo and in vitro. Her work is focused on characterizing these differences and understanding their implications for cell therapy and use in disease models. She is using a range of techniques and biocompatible engineered constructs to induce maturation of iPSC-CMs in vitro, such as micropatterning to induce anisotropic alignment in the culture, physical and electrical stimuli, elastic/ stretchable culture substrates to vary the mechanical load on the cells and co-culture with other cell types (e.g. fibroblasts). Dr Richard Jabbour, is a clinical cardiologist and BHF funded PhD research fellow, who is investigating the mechanisms of arrhythmia formation after iPSC-CM grafting by using a variety of platforms including engineered heart tissue (to manipulate the maturity of iPSC-CM), myocardial slices (to analyse iPSC-CM integration on a cellular level) and whether the use of biomaterials including conductive polymers or hydrogels can mitigate them.
The cellular electrophysiology group brings together different research approaches, from single cell to whole organ investigations. This multilayer approach is essential in the investigation of a chronic and progressive disease like heart failure. Complexity is essential in heart adaptability and explains the significant hurdles in defining the basis of heart failure and effective targets for treatment. A concerted effort within the lab and through our collaborations within the NHLI and external groups is required to tackle the difficult task of curing heart failure.