Contributions towards understanding the heart beat and cardiac myocyte action potential of the fruit fly

Studying the heart during normal function and disease is crucial to our understanding of human health and to aspects of animal biology from energy consumption and metabolism to lifespan. The heart of the fruit fly, Drosophila melanogaster, has emerged as a model system for studying heart function and particularly the linkage between genes, function, and physiology. Yet many aspects of D. melanogaster adult heart physiology remain unclear. Many of the insights from study of the D. melanogaster adult heart are obtained using a semi-intact preparation with artificial haemolymph, in which the beating heart tube is exposed by removing a fly’s head and thorax, and ventral abdomen. We used both in vivo electrophysiology and videography to investigate the heart-beat and cardiac myocyte action potential (AP) of the beating heart in the semi- intact preparation. This allowed the quantification of the heart-beat and AP, showing that there is considerable variation of both even in wild type flies. We show that variation in the heart-beat’s rate is, in part, due to transitions between the different types of heart-beat that the adult heart can generate and, in part, an artefact of the dissection itself. We also identify and describe at least two different patterns of cardiac myocyte AP in wild type flies: a single and a double-action potential. Together, these reveal an unappreciated variability in the adult heart and cardiac myocyte AP that has not previously been recognised or quantified, with implications of the uncritical use of the semi-intact preparation. At the commencement of the work towards this thesis, the major ion channels supporting the cardiac myocyte AP were unknown, though some channel-types had been identified. Using the semi-intact preparation, we investigated the voltage-gated ion channels and resulting currents that produce the cardiac myocyte AP using a range of Ca2+ and Na+ voltage-gated ion channel mutants, including classical mutations and RNAi lines. Using both videography and electrophysiology, we identify the voltage-gated a1 subunits Ca2+ encoded by the cacophony (cac) gene. Our evidence comes from changes in the frequency of the heart-beat in RNAi lines coupled with pharmacological evidence from application of the a1 subunits voltage-gated Ca2+ channel blocker, verapamil.We also show that forskolin, which upregulates the protein kinase A pathway, moderately increases heart-beat’s frequency. Despite the effects of cacophony RNAi on the heart beat’s frequency, cardiac myocyte APs were unaffected. We discuss how these results can be reconciled. Finally, we investigated the communication between cardiac myocytes through gap junctions, which is essential for propagation of the cardiac myocyte AP throughout the heart. We used carbenoxolone, a known connexin gap junction blocker, to block D. melanogaster cardiac myocyte gap junctions. This block was concentration dependent with an EC50 of 0.071 mM. Using intracellular recordings, we show that carbenoxolone is capable of increasing cardiac myocyte input resistance, consistent with blocking gap junctions. We also identify subthreshold electrical events in cardiac myocytes that are consistent with gap-junctional inputs from neighbouring cells. This emphasises the key role of gap junctions in the adult D. melanogaster heart and provides some of the first evidence that carbenoxolone is a blocker of innexin gap junctions. Taken together, the work in this thesis provides key insights into both the molecular and physiological mechanisms underpinning the adult D. melanogaster heart-beat, and the tools used by researchers to study these mechanisms.