Relationship between early and late events in the cardiac cycle as control points for therapeutic intervention

Heart failure is a major cause of disability and death worldwide, and approximately 50% of heart failure-related deaths are sudden and may be explained by abnormal electrical signals in the heart (arrhythmias). Heart failure patients have a 6- to 9-fold increased risk of sudden cardiac death compared to the general population. According to the "Heart of England screening study" the mortality risk for heart failure patients is 9% per year with a prevalence of ~6.6% of the population at 50 years of age (and which increases rapidly with age). In this study we will examine the interrelationships between electrical signals and calcium metabolism in heart cells with special emphasis on an integrative approach to understanding cell signalling. There is good evidence that heart failure is linked to changes in calcium signalling within cells which not only controls the force of contraction (and the ability of the heart to pump blood) but also affects electrical activity. The contraction of the heart, which is compromised in heart failure, is initiated by an electrical signal which causes calcium to be released inside the cell and it is the time course and amplitude of this calcium signal which is a major determinant of contraction force. However, the signal is not one way since calcium also affects electrical activity, especially later when the cell has to return its voltage to normal levels before the next beat. Our goal is to develop the scientific understanding needed to optimise both electrical and calcium signalling with drugs and/or by manipulation of key protein expression in the cell.

Using biophysical techniques, we will examine how the cell responds to measured changes in the expression of proteins that control heart cell voltage and how these changes affect the later electrical and calcium signalling events that occur in the cell. Using pharmacological agents we will dissect the electrical causes of normal and abnormal electrical activity together with how they are modulated by changes in calcium signalling. At the same time, we will probe how changes in voltage affect the calcium signalling mechanisms by using computer-generated electrical currents and feeding them into the cell to break feedback loops between calcium signalling and electrical currents to allow analysis. All of the data will be incorporated into computer models to allow us to re-integrate and test our understanding of how the electrical and calcium signalling changes work together to contribute to the disease state. At this point we will be able to identify how mixtures of drugs can be used to improve contraction strength while, at the same time, minimise arrhythmia risk.

It is also known that signalling pathways between the heart cell surface membrane and the intracellular store of calcium may become disrupted due to micro-anatomical changes in cell structure. It is not clear how heart cells adapt to these changes although we know that the intracellular calcium store release system becomes more 'leaky'. Using a novel form of illumination inside the cell (namely 2-photon excited flash photolysis) with special molecules that can react to the illumination, we will probe how microscopic elements of the calcium store respond to our artificially generated trigger signals. This will reveal the extent of this subcellular problem and therefore identify the utility of developing new therapeutic agents to ameliorate the leaky calcium store release. We will also be examining how subcellular signal transduction systems/pathways affect the integrated response of the system, to further refine possible points of control in the defective electrical and calcium signalling systems in the failing heart.

This integrative study aims to produce new insight into the complex interplay between early and late events in electrical excitation and Ca metabolism of healthy hearts and those in heart failure (HF). We will elucidate processes that lead to a reduced repolarization reserve in cardiomyocytes from an animal model chosen for similarity to humans. In particular we will examine: (1) the interplay of Ito with other currents and the effects of CaMKII and adrenergic stimulation on the time course of Ca release from the SR; (2) how modulation of early currents by drugs and expression changes impact later currents. We will develop drug combinations to limit early after-depolarization generation, control action potential (AP) duration and improve the uniformity of the Ca transient to improve cell contractile performance in HF.
Dynamic clamping will be used to measure AP responses to ionic current changes by injection or withdrawing currents generated by Hodgkin-Huxley model formulations. AP clamping will be used to directly measure repolarization reserve. Changes in ion channel expression in epi- and endo-cardial cells will be measured by qPCR and Western blotting. For both tubulated and detubulated regions of the cells (since failure also leads to t tubule loss), local RyR sensitivity will be probed by 3 dimensionally resolved 2-photon flash photolysis and measurement of release latency and its L-type Ca current dependence. This will be repeated in the presence of CaMKII stimulation, PKA stimulation for cells derived from both normal and failing hearts. Changes in location of proteins will be measured by immunocytochemistry and confocal microscopy. The interplay of subunit expression between ion channels will be examined by forced expression or knockdown of selected ion channel subunits in short term culture and IPSCs. Dose response curves for selected phamcological agents with be obtained in isolated myocytes and IPSCs.

Understanding how the heart works in health and disease is an important topic of great public and academic interest. The activity of cardiac ion channels and electrogenic transporters both underpins normal cardiac electrical and contractile function and, when altered in disease states including heart failure, contributes to morbidity and mortality. Heart failure affects three quarters of a million people in the UK, with the incidence likely to increase. Better understanding of the interaction between cardiac Ca2+ handling and ion channels involved in repolarization therefore offers new opportunities for rational drug design and, in particular, the development of new approaches to the treatment of arrhythmias and heart failure. This project is thus timely and should have major impact at both academic and social levels. To deliver that impact, we appreciate the importance of effective research is dissemination. The target audiences are academics, pharmaceutical industry, health professionals, schools and the wider public.
ACADEMICS: Electrophysiologists and those concerned with cardiac EC coupling, postdoctoral scientists employed on the project and the very wide range of undergraduate and postgraduate students benefit from this project. The state of the art methods being employed in the program will provide a rich training ground for young scientists at Bristol as well as help others address the biomedical problems they are working on through reading our Journal articles.
PHARMACEUTICAL INDUSTRY: Mutually beneficial collaboration with industry is an integral part of the way that we achieve our aims. Both applicants have a track record of industrial engagement/collaboration. JCH has previously collaborated with Pfizer in the area of cardiac safety pharmacology, whilst MBC has consulted on optical methods for live cell imaging. This track record will be exploited to commercialise and distribute, as appropriate, new intellectual property and tools developed during this project. Our initial work will involve the use of existing pharmacological tools. Where specific, commercially exploitable findings arise from their use, these will first be protected by the development of patents (including use patents), then exploited through industrial liaison. There is significant potential for novel approaches to the treatment of heart failure that do not exacerbate arrhythmia risk and our results are likely to have an impact in this area.
HEALTH PROFESSIONALS: Heart failure is common and current pharmacological treatments are limited in scope and efficacy. The increased understanding of mechanisms of repolarization reserve and early-after-depolarizations this programme will provide will aid development of rational approaches to arrhythmia treatment, whilst combination approaches involving transient outward current modulation may offer new possibilities for therapeutic exploitation. Therefore, the medical community will also benefit from any new developments in the field.
SCHOOLS: The future of science depends on enthusiastic young scientists. University open days provide opportunities for explaining science in a way that is accessible to prospective students and their parents. MBC already contribute to school science activities via the "Big Bang" program and our contributions are likely to increase as we increase our research profile in this important medical area.
THE WIDER PUBLIC: Both applicants are committed to engage public interest and to shape public perception about the benefits of scientific discovery. We will take every opportunity to directly engage the public and schools through initiatives coordinated by the Bristol University Centre for Public Engagement, Bristol Heart Institute and by local attractions (e.g. the science activity centre '@Bristol'). Our published findings will be promoted to the public through the Bristol Heart Institute, University web sites and the University Press Office.