2Cardiac Electrophysiology Group, Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, England
3Scientific Department, Biobide, San Sebastian, Gipuzkoa, Spain
4Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA
5Development and Aging Program, Neuroscience, Aging, and Stem Cell Research Center, Burnham Institute for Medical Research, La Jolla, CA, USA
6Center of Regenerative Medicine in Barcelona, Barcelona, Spain
7the Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada
A new method for detection and quantification of heartbeat parameters in Drosophila, zebrafish, and embryonic mouse hearts
High speed movie of a 3-wk-old fly heart that was originally captured at 146 frames per second and then exported to a 20s movie clip.
Movement is detected by monitoring changes in intensity of individual pixels from frame to frame in each movie. This movie represents the first 10 s of Movie 1; pixels that show significant changes in intensity are highlighted in red. This movie is a 1/4 speed version of Movie 1.
Immobilized 3-d-old zebrafish larva were filmed as described for Drosophila hearts at 10× magnification. Anterior is to the left of the frame and ventral is up. Ventriclar contractions are very prominent in this orientation.
Eight-day-old mouse embryos were surgically removed following cervical dislocation of the mother, were cultured for 1 h in oxygenated DMEM at 37oC, and then filmed as described for Drosophila hearts at 10× magnification. Anterior is to the left and dorsal is up; the movie was originally captured at 146 frames per second and the clip is 10 s long.
The genetic basis of heart development is remarkably conserved from Drosophila to mammals, and insights from flies have greatly informed our understanding of vertebrate heart development. Recent evidence suggests that many aspects of heart function are also conserved and the genes involved in heart development also play roles in adult heart function. We have developed a Drosophila heart preparation and movement analysis algorithm that allows quantification of functional parameters. Our methodology combines high-speed optical recording of beating hearts with a robust, semi-automated analysis to accurately detect and quantify, on a beat-to-beat basis, not only heart rate but also diastolic and systolic intervals, systolic and diastolic diameters, percent fractional shortening, contraction wave velocity, and cardiac arrhythmicity. Here, we present a detailed analysis of hearts from adult Drosophila, 2–3-day-old zebrafish larva, and 8-day-old mouse embryos, indicating that our methodology is potentially applicable to an array of biological models. We detect progressive age-related changes in fly hearts as well as subtle but distinct cardiac deficits in Tbx5 heterozygote mutant zebrafish. Our methodology for quantifying cardiac function in these genetically tractable model systems should provide valuable insights into the genetics of heart function.
The fruit fly, Drosophila, is a powerful genetic model system with a multitude of tools for manipulating genes and gene expression. This system has provided valuable insight into the cellular mechanism underlying heart development in the fly and this information has led to the identification of key regulators of heart development in vertebrates. Several groups have begun to examine heart function in the fruit fly with the goal of using this system as a physiological model that can be manipulated genetically (1-8). Genetic manipulations of ion channel genes in Drosophila, including L-type Ca2+ channels and several types of K+ channels, suggest that the currents contributing to heart function in flies are remarkably similar to those in human hearts (3,8-11). Several mutations produce effects in the fly that mimic human heart disease syndromes (3,5) reviewed in References 12-14,. Thus, the fly heart will be useful as a myocardial model of human heart disease.
A number of methods have been developed to detect and quantify heart rate in fruit flies. Manual counting of heart beats visualized through the cuticle of intact pupae and flies has been used previously, but is not practical or accurate for long periods of time or during periods of high-frequency beating. Heart rates have also been obtained by manual counting from slow motion replay of videotape recordings (2), which provides a more accurate determination of rate but does not provide information about the relative lengths of diastole and systole, which are important parameters for detailed heart beat analysis. Automated detection of light intensity changes has also been used to provide an objective measure of heart rate (10,11,15-21), as has edge tracing (6), but again, these methods primarily provide information about rate. Electrical recordings (22) can also be used to provide heart rate information, but obtaining stable recordings from beating hearts is technically difficult even for short periods of time. Finally, optical coherence tomography (OCT) (7) has been employed to obtain a number of heart beat parameters, including diastolic and systolic intervals, but this method requires highly specialized equipment, tracks heart activity for only limited periods of time, has limited spatial resolution, and requires manual calculations of a limited numbers of beats from M-mode records. Thus, it is desirable to standardize and automate a method for obtaining reliable measurements of the dynamic parameters of heart function for large samples of individuals over significant periods of time and for all the heart beats in a record.
We have developed a methodology for analyzing a number of contraction-relaxation parameters in the myogenic heart of Drosophila that is also applicable to other model systems. Our method combines a denervated, exposed fly heart with a unique set of movement detection algorithms that automatically and precisely detect and measure beat-to-beat contraction parameters captured in low or high speed movies providing both analytical and statistical power. The output provides detailed information concerning pacemaker activity and contraction-relaxation parameters including heart rate, systolic intervals (SI) and diastolic intervals (DI), systolic and diastolic diameters, contraction strength, heart rhythmicity, and contraction wave velocity (CWV) along the heart tube. We have successfully used these movement detection algorithms to quantify heart beat parameters in Drosophila, as well as larval zebrafish and embryonic mouse hearts.Materials and methods Semi-intact Drosophila heart preparation
Two wild-type laboratory strains of Drosophilia (yw and w1118) were maintained and aged as described previously (3,6). Abdominal heart tubes were exposed by cutting off the head and ventral thorax of the fly and then removing the ventral abdominal cuticle and all internal organs. Dissections were performed under an artificial adult hemolymph (based on References 23, and 24) containing 108 mM NaCl2, 5 mM KCl, 2 mM CaCl2, 8 mM MgCl2,1 mM NaH2PO4, 4 mM NaHCO3, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM sucrose, and 5 mM trehalose, at pH 7.1. Recordings of heart activity were acquired from semi-intact Drosophila preparations at room temperature using a Hamamatsu EM-CCD digital camera (McBain Instruments, Chatsworth, CA, USA) mounted on a Leica DM-LFSA microscope with a 10× water immersion lens (McBain Instruments) and Simple PCI image capture software (Compix Imaging System, Selwicky, PA, USA). Frame rates were 100–150 fps; all movies were 60 s in length. See Supplementary Movie 1 (available at www.BioTechniques.com) for an example.