Cell Electrophysiology
Dr Cesare Terracciano, Head of Group
Heart failure is a progressive disease and, when severe, has a prognosis similar to the most malignant forms of cancer. In Europe and the United States the disease has an average prevalence of 2.5% in the population. The only long-term, effective therapeutic option for patients with end-stage heart failure is cardiac transplantation but this is inadequate, due to the small number of available donor organs.
In the last few years the concept that ventricular remodelling produced by heart failure is irreversible has been challenged. Different strategies, including mechanical unloading, gene and cell therapy, at least in certain conditions, have shown to prevent or reverse myocardial remodelling and induce myocardial functional improvement. However, the mechanisms involved in this improvement are unknown. Our major objective is to determine the mechanisms that bring about myocardial functional improvement after mechanical unloading, gene and cell therapy. In particular, we are interested in the ion channel physiology and cytoplasmic calcium regulation changes . We are also interested in determining the cell-to-cell communication properties that may be the basis for arrhythmic disorders in heart failure and a possible target for therapy. The techniques we employ are single cell current- and voltage-clamping using sharp electrodes and patch-clamping techniques, dual patch-clamping, fluorescence and confocal microscopy in conjunction with the use of intracellular [Ca], [Na] and pH-sensitive dyes, assessment of cell and sarcomere shortening, and electrophysiology of cell monolayers using multielectrode arrays (see attached movie) and optical mapping.
Manipulation of mechanical load as a platform for the treatment of heart failure - Bridge-to-Revovery
Ventricular assist devices (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’s structure and function. 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 also 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 etiology- dependent. During the course of VAD treatment, mechanical unloading may be beneficial, inducing reverse remodeling, but may also have 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 remodeling of the failing ventricle. We were 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).
T-tubular structure, cytoskeleton and cardiac function
Another important aspect of our research is the study of the role of changes to cellular cytoarchitecture in disease and after therapeutic interventions. In particular, we have investigated the role of changes to structure in altering the electrophysiology of cardiac cells. We discovered a previously undescribed role for changes to one of the cytoskeletal proteins, protein 4.1, in changing the electrophysiology of cells with possible roles in disease (Stagg et al., Circulation Research 2008). In collaboration with Professor Thomas Brand at the Harefield Heart Science Centre, we are currently investigating the role of another family of accessory proteins belonging to the Popeye family, which may be implicated in disturbances of the heart rhythm.
Another important area of investigation in our laboratory is the role of alterations in the transverse (t)-tubule structure in response to changes in load (Ibrahim et al., FASEB Journal 2010). The 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). 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. We are investigating the mechanisms mediating these changes with a view to applying them in the context of heart failure.
Human Pluripotent Stem Cells: characterization and strategies for maturation.
The heart as adult cardiac tissue has limited regenerative capacity and this is particularly problematic following an injury of the heart, such as during a heart attack. The cardiac tissue cannot reform normally, leading to heart failure. Consequently, there has been significant interest in investigating the potential of stem cells from different sources to replace damaged cardiomyocytes. Several cell types have been investigated for this purpose. Pluripotent stem cells are of significant interest as they are of human origin and can be differentiated in every tissue in the body, including the heart. In particular, induced pluripotent stem cells (iPSC), which are derived from adult, not embryonic cells, are interesting.
IPSC, unlike embryonic stem cells, do not require the ethically problematic destruction of embryos and as allogeneic transplantation is feasible host immune rejection can potentially be avoided in cell therapy. Furthermore, as iPSC can be generated from somatic cells of patients with specific disease phenotypes it is theoretically possible to create in vitro disease models using iPSC derived cardiomyocytes.
The structure and function of cardiomyocytes differentiated from pluripotent stem cells in-vitro differs significant from adult cardiomyoctes. Our work is concerned with characterizing these differences and understanding their implications for cell therapy and use in disease models. We employ a range of techniques to manipulate the cells and are interested in their alignment and load.
Human heart slices - a novel multicellular system suitable for physiological and pharmacological studies:
Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human myocardium. We have developed and validated a new multicellular preparation with preserved native structure and function that can be easily prepared from human heart biopsies (Camelliti et al., JMCC 2011). Thin vibratome-cut myocardial slices are prepared from left ventricular transmural biopsies obtained from end-stage heart failure patients undergoing heart transplant or ventricular assist device implantation. We have assessed contractile and electrical activity using multi-electrode arrays and optical mapping and performed metabolic analysis using high performance liquid chromatography/mass spectrometry. Morphology and gap junction distribution were evaluated with histology and confocal microscopy. We found that regular contractility can be observed at a range of stimulation frequencies (0.1-1Hz) and stable electrical activity is maintained for at least 8 hours from slice preparation. Slices stimulated along the direction of the myocardial fibres (Fig A, blue arrow and B) have a higher conduction velocity compared with slices stimulated transversally (Fig A, red arrow and C). The ATP/ADP and phosphocreatine/creatine ratios are comparable to intact organ values. Tissue architecture and gap junction distribution are representative of native myocardium. Our results suggest that viable myocardial slices with preserved structural, biochemical and electrophysiological properties can be prepared from adult human heart biopsies and offer a novel preparation suitable for the study of heart failure and drug screening. This project is led by Dr Patrizia Camelliti (link) who is an Imperial College Research Fellow.
Selected publications:
Camelliti P, Al-Saud SA, Smolenski RT, Al-Ayoubi S, Bussek A, Wettwer E, Banner NR, Bowles CT, Yacoub MH, Terracciano CM. Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies.J Mol Cell Cardiol. 2011 Sep;51(3):390-8.
Ibrahim M, Al Masri A, Navaratnarajah M, Siedlecka U, Soppa GK, Moshkov A, Al-Saud SA, Gorelik J, Yacoub MH, Terracciano CM.Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface. FASEB J. 2010 Sep;24(9):3321-9.
Terracciano CM, Miller LW, Yacoub MH.Contemporary use of ventricular assist devices. Annu Rev Med. 2010;61:255-70.
Chambers JC, Zhao J, Terracciano CM, Bezzina CR, Zhang W, Kaba R, Navaratnarajah M, Lotlikar A, Sehmi JS, Kooner MK, Deng G, Siedlecka U, Parasramka S, El-Hamamsy I, Wass MN, Dekker LR, de Jong JS, Sternberg MJ, McKenna W, Severs NJ, de Silva R, Wilde AA, Anand P, Yacoub M, Scott J, Elliott P, Wood JN, Kooner JS. Genetic variation in SCN10A influences cardiac conduction. Nat Genet. 2010 Feb;42(2):149-52.
Stagg MA, Carter E, Sohrabi N, Siedlecka U, Soppa GK, Mead F, Mohandas N, Taylor-Harris P, Baines A, Bennett P, Yacoub MH, Pinder JC, Terracciano CM. Cytoskeletal protein 4.1R affects repolarization and regulates calcium handling in the heart.Circ Res. 2008 Oct 10;103(8):855-63.
