Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of lightsensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of lightsensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Optogenetics allows non-invasive control of cardiac function by inducing physiological heart distress. The main challenge for optogenetic pacing is relatively shallow light penetration into biological tissue. Recently, ChRmine was reported as an excitatory opsin that can respond to much lower light power which induces a larger cellular membrane electrical current than the previously characterized opsins. It has been shown that ChRmine expressed deep inside the mouse brain (~7 mm) sensed the external illumination leading to mouse behavior change. However, the ChRmine application for optogenetic cardiac function control has not been optimized yet. This study demonstrates the feasibility of ChRmine-mediated restorable tachycardiac pacing in Drosophila (fruit fly) hearts non-invasively and discusses the influence of different parameters of both optical settings and gene expression levels to achieve its optimal performance. custom optical coherence microscopy (OCM) was synchronized with the illumination module to monitor the dynamics of the fly heart while light pulses were applied. The quantification of cardiac function was performed by lab-developed software FlyNet 2.0+. The results demonstrate successful pacing at designed frequencies higher than the resting heart rate in ChRmine flies. Irradiance power density and pulse width were tested for minimal light power input. Illumination protocols were set up to provide pacing control. Different transcriptional activator strains were crossed in to enhance transgenic ChRmine expression. Opsin ligand concentration in Drosophila media was adjusted for saturating opsin membrane channel opening. Red light wavelengths (617 or 656 nm) were considered to promote penetration depth for optogenetic activation. This study will serve as practical guidance for performing optogenetic pacing using ChRmine opsin.