Development of a platform for the assessment of cardioactive compounds by quantitative and simultaneous analysis of subcellular cAMP signalling and cardiac myocyte electro-mechanical effects
LEAD SUPERVISOR: Prof Manuela Zaccolo, Department of Physiology Anatomy and Genetics
Co-supervisor: Dr Jakub Tomek, Department of Physiology Anatomy and Genetics
Commercial partner: InoCardia Ltd, Coventry
cAMP signalling is central to sympathetic regulation of heart rate and contractility and is altered in heart disease. The success of therapeutics to treat cardiac disease, such as heart failure, inevitably depends on how the drug impacts on this pathway.
Recent research, including from the Zaccolo’s laboratory, established that cAMP is compartmentalised in multiple, co-existing subcellular domains. In addition to β-adrenergic receptor stimulation, cAMP is generated in response to activation of multiple other G-protein coupled receptors (GPCR), affecting many other cardiac myocyte activities (energy metabolism, gene expression, cell survival and others), some with potential deleterious effects on function (e.g. cardiac remodelling, apoptosis). The multiple cAMP signals generated by GPCRs in different subcellular compartments must be tightly regulated locally to ensure correct execution of sympathetic control.
How current treatments for heart disease impact subcellular cAMP signalling is almost completely unexplored and drug discovery pipelines largely assess the effect of lead compounds on sympathetic signalling by measuring global cAMP levels in cell population lysates. However, it is becoming increasingly clear that global changes in cAMP do not dictate functional outcome, which rather depends on the precise combination of cAMP changes at distinct subcellular locations. A detailed understanding of how drugs impact on local cAMP levels is key to maximise therapeutic benefit and minimise side effects. The compartmentalisation of cAMP provides a unique opportunity for developing precision therapy approaches at the subcellular level, where bioactive molecules can target an individual subcellular domain without affecting neighbouring subcellular sites.
The overall aim of this project is to develop a tool to predict the functional outcome of new compounds by determining their impact on subcellular cAMP domains. For this purpose, we will investigate how current drugs, with well-established inotropic effects, impact subcellular local cAMP levels and we will develop a robust, quantitative approach to link local subcellular cAMP changes with their effect on contractility.
The project will bring together state-of-the art imaging using genetically encoded, FRET-based reporters (developed by the Zaccolo’s laboratory) and a robust and highly sensitive cardiomyocyte contractility Work-Loop model that predicts drug-induced inotropy and electromechanical coupling alterations, developed by the industrial partner, InoCardia Ltd.
The new platform will enable simultaneous measurement, in the same cardiomyocyte, of a variety of parameters that relate to force of contraction, encompassing the utilisation of the proprietary InoCardia Work-Loop model, and local changes in cAMP measured with targeted FRET reporters. The cAMP sensors will be targeted to specific subcellular sites (e.g. plasmalemma, myofilaments, sarcoplasmic reticulum, nucleus, mitochondria) and will provide quantitative information on subcellular cAMP concentrations with high spatial and temporal resolution. Detection of local cAMP changes provides a more direct readout and mechanistic insight compared to other available readouts (e.g. calcium transient, action potential) which are downstream of adrenergic signalling but result from more complex molecular events. The new system will be validated by investigating how drugs with well-established inotropic effects (e.g. β-agonists, β-blockers, phosphodiesterase inhibitors etc.) impact subcellular cAMP pools and how this affects electro-mechanical properties. Biased agonism/antagonism will also be explored.
Apply using course: DPhil in Physiology, Anatomy and Genetics
