Luciferase Expression for ATP Imaging: Application to Cardiac Myocytes

Abstract

Targeted luciferase is a powerful tool for measuring adenosine triphosphate (ATP) levels within various compartments of living cells. This chapter focuses on some potential applications of this system, with particular reference to the measurement of mitochondrial and cytosolic ATP changes in working cardiac myocytes. The chapter presents experiments demonstrating that adenovirally expressed luciferase is a useful tool for measuring changes in ATP in different compartments of living primary cells such as single rat cardiac myocytes. Although clear changes in ATP levels could be observed on large changes in workload in these cells, it can be concluded that in normal circumstances ATP generation is extremely well matched to demand, in particular that ATP levels in the cytosol are extremely well buffered. The relative ATP concentration within individual cell compartments can be estimated using a bulk approach, where luminescence from mammalian cells expressing the targeted luciferase reporters is measured in a luminometer. The chapter tests this approach by expressing various targeted recombinant luciferases by standard transient transfections with plasmid vectors.