Myocardial slices to study cardiovascular disease in vitro

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

The use of living animals in cardiovascular research is currently necessary due to the lack of appropriate and relevant in vitro models. Current in vitro models suffer from being reductionists (cell lines and stem cell-derived cardiac cells, often immature, no 3D structure) and/or are subjected to rapid changes induced by culture (can only be used for acute studies). The development of the living myocardial slice (LMS) model with its preserved tissue structure and ability to remain stable in culture for several days/months, therefore has the potential to have a significant impact upon the 3Rs. Several slices can be prepared from the same heart so that, for instance, 1 rat heart used in LMSs experiments can provide the same number of experimental repeats of 6-8 rat hearts used for experiments with other preparations. In addition, LMSs are ideal to study both structure and function and, with one LMS, one can perform several experiments compared with other models which need several hearts. Because slices are prepared by pure mechanical dissociation, the same slices analysed for contractility experiments can then be dissected in smaller slices and used for biochemistry, imaging and other studies. This ability to optimise the use of tissue can bring about a minimum of 50% reduction of the number of animals needed.

In our project we will test the feasibility to induce injury on slices in vitro and not in the living animals. This aspect is likely to have a profound impact on cardiovascular research in many academic labs and industry. The ability to induce disease "in a dish" means that laboratories can avoid performing severe regulated procedures aimed at inducing cardiovascular disease in living animals.

Furthermore, by using the LMS model it is now possible to optimise the use of available human heart tissue. While this is limited, transplant centres perform approximately 20-30 heart transplants per year. Another expanding source of human heart tissue is the implantation of left ventricular assist devices (LVADs), now performed routinely in many cardiac surgery centres (20-30 patients per year/per centre). The implantation of LVADs is accompanied by the removal of a core of ventricular tissue that is amenable to the preparation of the LMS model. In addition, unused donor human hearts are available through dedicated research programmes, making the use of human LMSs not unrealistic and particularly relevant.

We will test several kinds of disease in vitro using LMSs, and we will validate the use of this technique to study the effects of drugs that are toxic for the heart (some treatments for cancer bring about cardiac disease) and drugs that can be used to treat cardiac disease. Overall, we expect that the method developed by this study will became a major tool in cardiovascular research with important implications for the use of living animals in research.

Technical Summary

Living myocardial slices (LMSs) are a new in vitro model of intermediate complexity and with the great potential to optimise and reduce the use of living animals in cardiovascular research. We have recently obtained methods that prevent or substantially reduce culture-induced remodelling of LMSs for several days. In this project, we will test the hypothesis that LMSs kept in stable conditions for several days can be subjected to relevant injury in vitro, and can be used to reduce/replace animals in cardiovascular research. We will prepare slices from rat and rabbit hearts without subjecting the animals to regulated procedures and human heart biopsies obtained from heart transplant programmes. We will apply cryoinjury to LMSs in culture, to induce localised and reproducible myocardial necrosis, induce global ischemia/reperfusion, and mechanically overload the LMSs using electromechanical simulation protocols involving high preload/afterload, to simulate the processes that lead to heart failure. From the resulting injured LMSs, we will measure contractility, parameters of calcium regulation, tissue structure, ultrastructure of cardiac cells and biochemical assays already established in our laboratory. We will validate the effects of toxic substances (antineoplastic drugs with cardiotoxicity, e.g. sunitinib) and beneficial treatments (ACE-inhibitors, beta-blockers, ivabradine amongst others) on the contractile and electrical properties of the model. The validated LMSs models of injury will have a clear role in the cardiovascular research pipeline, bridging the gap between in vitro and in vivo models, with the potential to substantially reduce the number of, and/or partially replace, living animals undergoing high severity regulated procedures and utilised in cardiovascular discovery.

Publications

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