THE ZEBRAFISH AS A MODEL FOR STUDIES OF CARDIAC MECHANO-ELECTRIC FUNCTION

Abstract

Factors affecting cardiac function include those external to the heart (e.g., the autonomic nervous system, ANS) and intrinsic processes that allow rapid beat-by-beat adaptation to change in physiological demands. The overall objective of my PhD research was to develop and utilise methods to investigate the integrated control of cardiac electrical and mechanical activity by intrinsic and extrinsic mechanisms in the zebrafish. This involved: i) creation of a micro-cannulation technique to acutely manipulate the heart’s hemodynamic load in vivo in intact zebrafish larvae and ii) intracellular microelectrode measurements of cellular electrical activity in situ in isolated zebrafish hearts combined with its optogenetic manipulation. In the developing heart, effects of acute changes in hemodynamic load on cardiac function are not well established but may be critical for the pre-neuronal control of cardiac excitation and contraction. The specific aims of the primary portion of my thesis were to determine the influence of acute changes in hemodynamic load on cardiac function in vivo in intact zebrafish larvae during: i) different stages of cardiac development, to define its evolution in the developing heart and ii) pharmacological interrogation of the ANS and stretch-activated channels, to determine intrinsic and extrinsic mechanisms driving observed functional changes, along with measurement of membrane potential and intracellular calcium-dynamics using genetically-expressed fluorescent reporters. It was found that an acute increase in hemodynamic load at 2 days post fertilisation (dpf) causes a small decrease in heart rate, which becomes an increase by 6 dpf and is further increased at 14 dpf, while stroke volume was increased at all developmental stages. Interventions indicated that effects on cardiac rhythm are mediated by both intrinsic (stretch) and extrinsic (ANS) mechanisms, while the effects on stroke volume are purely intrinsic. Metrics of intracellular calcium handling (time to peak transient, transient duration) were altered with acute hemodynamic loading, while voltage dynamics remained unchanged. Activation of cation-conducting channelrhodopsins leads to membrane depolarisation, allowing the effective triggering of action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarisation-induced inhibition of AP generation has remained challenging. The green-light activated channelrhodopsin GtACR1 mediates chloride-driven photocurrents that have been shown to silence AP generation in neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocytes activity. The aim of this project was to determine the effects of GtACR1 with pulsed and sustained light stimulation in intact zebrafish hearts. It was found that both modes of light stimulation resulted in cell depolarisation, such that pulsed light paced the heart, and sustained light caused silencing. While this does not address the need for optogenetic silencing by hyperpolarization, GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarisation. Overall, my research has revealed factors involved in the regulation of cardiac function in response to acute changes in hemodynamic load during development and has shed light on the potential use of optogenetics for cardiac control. My novel findings may help us toward a better understanding of disturbances that arise in congenital heart disease during cardiac development and maturation and how they may ultimately be treated.