A new method for detection and quantification of heartbeat parameters in Drosophila, zebrafish, and embryonic mouse hearts

Abstract

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.

Figure 1. Systolic and diastolic heart diameters

(A) Movie still of three segments in the fly abdomen showing the exposed heart during diastole. The position of the heart walls is indicated by white arrowheads, and circles indicate positions marked by the user and used by the program to calculate heart diameters. The vertical dashed lines show the orientation of pixel strips used to generate the M-modes shown in Figure 2. (B) The same fly heart during systole; arrowheads mark the changed location of the heart tube edge during contraction. (C) Quantification of the diastolic and systolic diameter measurements for yw and w¹¹¹⁸ laboratory wild-type strains of Drosophila. Data points represent the mean (±SEM) for 17–30 flies per data point. The decrease in diastolic diameter shows a significant age dependence (ANCOVA, P = 0.003) but the systolic diameter decrease is not significant in this study (ANCOVA, P = 0.08). (square, yw strain; triangle, w¹¹¹⁸ strain. Black lines represent diastole, gray lines represent systole) (D) Percent fractional shortening (% FS) provides an estimate of the ejection volume and is obtained from the data shown in C (see Equation 2). The decrease of % FS with age is significant (ANCOVA, P = 0.04) and is due to the relatively greater decrease of diastolic diameter as compared to systolic diameter. (square, yw strain; triangle, w¹¹¹⁸ strain).

Figure 2. Movement detection from high-speed digital movies

(A) Movement detection for a 1-week-old fly using the Frame Brightness Algorithm; peaks indicate individual heart contractions. (B) Movement detection for a 1-week-old fly using the Changing Pixel Intensity Algorithm. For each beat the first peak represents the movement due to contraction, whereas the second (or additional) movement is due to relaxation. The number of frames for each detected diastolic interval is printed above the horizontal line which represents the movement threshold. (C) M-mode generated by electronically excising and aligning 1-pixel-wide, vertical strips from successive movie frames. These strips span the heart tube and are taken from the same location in each frame of the movie (approximately the middle of the third abdominal segment; see dashed lines in Figure 1, A and B). Thus, M-modes show movement of the heart tube edges (in the vertical y-axis) over time (on the x-axis). The movie frames analyzed in A and B were used to produce the M-modes shown in C (note alignment of movie frame numbers on the x-axis for panels A–C and D–F); 200 frames represents ∼1.5 s. (D) Movement detection using the Frame Brightness Algorithm for a 7-week-old fly. (E) Movement detection using the Changing Pixel Intensity Algorithm for a 7-week-old fly, note incomplete relaxations/non-sustained fibrillations. (Solid line represents the movement threshold and detected diastolic intervals.) (F) M-mode from the same 7-week-old fly movie.

Figure 3. Heart beat intervals and quantification of arrhythmicity

(A) Movement trace showing DI detection (horizontal lines between movement peaks). Duration of the DI is given as the number of frames between successive movement traces; note the regularity of DI in this young (1-week-old) fly. (B) Movement trace from an old fly (5 weeks) showing increased irregularity of both systolic and diastolic interval lengths. (C) Changes in heart period with age (Mean ± SEM, 17–30 flies per data point). (D) Diastolic intervals (black lines) and systolic intervals (gray lines) showed significant increases as a function of age (Mean ± SEM, 17–30 flies/data point). (E) “Arrhythmicity Index” (AI) calculated as the heart period standard deviation normalized to the median heart period. Data points represent the average AI for all the flies in each age group (17–30 flies per data point). The age-dependent increase in AI is significant and reflects the observed increase in arrhythmic events that occurs as flies age (shown qualitatively in Figure 2C). For C–E, age-dependent changes in cardiac parameters were modeled hierarchically using ANCOVA, ANOVA for genotype as a function of each parameter and the t-tests were employed to determine if there were significant differences between yw and w¹¹¹⁸ and between young (1-week-old) and old flies [3–7 weeks, *P < 0.05; see also (3)]. Square, yw strain; triangle, w¹¹¹⁸ strain.

Figure 4. Zebrafish heart parameters

(A) 10-s M-modes from movies of 2-3-day-old zebrafish hearts. Wild-type M-modes (top trace) show regular heart contractions as evidenced by the significant movement of the heart edge in the dorsal region of the heart (bottom edge trace in the m-mode). All Tbx5 heterozygotes (lower traces) showed aberrations in their M-modes, primarily characterized by more prolonged contractions with heart wall movements that were noticeably less robust and less fluid. (B) Contraction traces and corresponding M-mode from a wild-type zebrafish showing the correlation between the Changing Pixel Intensity Algorithm and the movements of the heart wall. (C) Heart period was measured as the interval between the start of one diastole and the beginning of the next. Heart periods were measured for every beat in each movie and averaged for each fish. Results for 16 wild-type and 27 Tbx5 heterozygote zebrafish show a significant increase in the heart period in heterozygotes compared with controls (*P = 7 × 10^-5). (D) Percent fractional area change (% FAC) was measured as the percent change in the ventricular surface area between diastole and systole. Heart measurements were made only if all heart edges were clearly visible in the movie frames. Results from 15 wild-type and 12 Tbx5 heterozygotes show a significant reduction in the % FAC in heterozygotes compared with controls (*P = 0.02). (E) A comparison of the ventricular surface areas of hearts measured in (D) during diastole and systole indicate that the decrease in % FAC is due to a decrease in the diastolic size of the hearts. Diastolic surface area of Tbx5 heterozygotes was significantly smaller than controls whereas systolic surface area did not differ significantly between the two groups (*P = 0.009). For C–E, results are given as mean ±SD. * indicates difference is significant based on unpaired, two-tailed t-test.