A method to measure myocardial calcium handling in adult Drosophila

Abstract

Rationale: Normal cardiac physiology requires highly regulated cytosolic Ca(2+) concentrations and abnormalities in Ca(2+) handling are associated with heart failure. The majority of approaches to identifying the components that regulate intracellular Ca(2+) dynamics rely on cells in culture, mouse models, and human samples. However, a genetically robust system for unbiased screens of mutations that affect Ca(2+) handling remains a challenge.

Objective: We sought to develop a new method to measure myocardial Ca(2+) cycling in adult Drosophila and determine whether cardiomyopathic fly hearts recapitulate aspects of diseased mammalian myocardium.

Methods and results: Using engineered transgenic Drosophila that have cardiac-specific expression of Ca(2+)-sensing fluorescent protein, GCaMP2, we developed methods to measure parameters associated with myocardial Ca(2+) handling. The following key observations were identified: (1) Control w(1118) Drosophila hearts have readily measureable Ca(2+)-dependent fluorescent signals that are dependent on L-type Ca(2+) channels and SR Ca(2+) stores and originate from rostral and caudal pacemakers. (2) A fly mutant, held-up(2) (hdp(2)), that has a point mutation in troponin I and has a dilated cardiomyopathic phenotype demonstrates abnormalities in myocardial Ca(2+) handling that include increases in the duration of the 50% rise in intensity to peak intensity, the half-time of fluorescence decline from peak, the full duration at half-maximal intensity, and decreases in the linear slope of decay from 80% to 20% intensity decay. (3) Hearts from hdp(2) mutants had reductions in caffeine-induced Ca(2+) increases and reductions in ryanodine receptor (RyR) without changes in L-type Ca(2+) channel transcripts in comparison with w(1118).

Conclusions: Our results show that the cardiac-specific expression of GCaMP2 provides a means of characterizing propagating Ca(2+) transients in adult fly hearts. Moreover, the adult fruit fly heart recapitulates several aspects of Ca(2+) regulation observed in mammalian myocardium. A mutation in Drosophila that causes an enlarged cardiac chamber and impaired contractile function is associated with abnormalities in the cytosolic Ca(2+) transient as well as changes in transcript levels of proteins associated with Ca(2+) handling. This new methodology has the potential to permit an examination of evolutionarily conserved myocardial Ca(2+)-handing mechanisms by applying the vast resources available in the fly genomics community to conduct genetic screens to identify new genes involved in generated Ca(2+) transients and arrhythmias.

Figure 1. Measurement of Ca²⁺-dependent fluorescence in adult hearts from w¹¹¹⁸; tinC-GCaMP2

A. Representative bright field image (top) and fluorescence image (bottom) of the heart prepared from an adult w¹¹¹⁸; tinC-GCaMP2. The heart preparation includes the dorsal cuticle and is oriented with abdominal segments (A1, A2, A3, and A4) as shown. B. Representative pseuodocolored isochrone map from a w¹¹¹⁸; tinC-GCaMP2 heart showing caudal to rostral Ca²⁺ wave propagation. Blue and red pixels indicate the earliest and latest time points during the recording, respectively. The pseudocolored line represents the pixel intensities averaged along the vertical columns. C. Fluorescence intensity traces for three individual pixels representing the earliest (blue), middle (green), and latest (red) times during the Ca²⁺ wave propagation and the superimposed traces are shown. A 100 msec time bar is shown.

Figure 2. Determination of Ca²⁺ propagation in adult hearts from w¹¹¹⁸; tinC-GCaMP2

A. The abdominal A2 and A3 segments from a representative w¹¹¹⁸; tinC-GCaMP2 heart in the rostral to caudal orientation is shown. B. Three different representative temporal isochrones with blue and red pixels indicating the earliest and latest times of activation, respectively, are shown (top). Series of consecutive 5 msec frames from temporal isochrone movies are shown. Caudal to rostral (left column), rostral to caudal (middle column), and bidirectional (right column) Ca²⁺ propagation with the corresponding percentages from 714 measures in 32 w¹¹¹⁸; tinC-GCaMP2 hearts are shown. C. Individual pixel intensity traces along the heart from w¹¹¹⁸; tinC-GCaMP2 showing caudal to rostral Ca²⁺ propagation during a 600 msec recording. D. Individual pixel intensity traces along the heart from w¹¹¹⁸; tinC-GCaMP2 showing rostral to caudal Ca²⁺ propagation during a 600 msec recording. E. Individual pixel intensity traces along the heart from w¹¹¹⁸; tinC-GCaMP2 showing bidirectional Ca²⁺ propagation originating from both the rostral and caudal regions during a 600 msec recording.

Figure 3. Comparison of anterograde and retrograde directed Ca²⁺ transients in adult hearts from w¹¹¹⁸; tinC-GCaMP2

A. Representative trace of averaged pixel intensities from a w¹¹¹⁸; tinC-GCaMP2 heart. Definitions of dF/dt_max (A), the duration of the 50% rise to peak fluorescence intensity (B), the duration of the peak to 50% decay in fluorescence intensity (C), FDMH (D), and the slope of linear decay from 80% to 20% fluorescence intensity (E) are shown. B. Comparison between parameters of pixel intensity traces and conduction velocity for anterograde (i.e.; caudal to rostral) (n=25) and retrograde (i.e.; rostral to caudal) (n=31) Ca²⁺ transients in w¹¹¹⁸; tinC-GCaMP2 hearts. There were no statistically significant differences in the parameters measured between anterograde and retrograde Ca²⁺ wave propagations.

Figure 4. Ca²⁺ transients in adult hearts from w¹¹¹⁸; tinC-GCaMP2 are dependent on extracellular Na⁺, extracellular Ca²⁺ and intracellular Ca²⁺ stores

A. Representative trace of the generated Ca²⁺ transients in w¹¹¹⁸; tinC-GCaMP2 in buffer containing 110 mM [Na⁺]_o and after exposure to 2 mM [Na⁺]_o buffer. Two second segments of the time course are shown. Inset table shows fold changes in transient rate and conduction velocity after exposure to 2 mM [Na⁺]_o. (*p<0.05 by paired t-test, n=9). B. Representative trace of generated Ca²⁺ transients in w¹¹¹⁸; tinC-GCaMP2 before and after the administration of 5 mM EGTA. C–E. Representative serial traces of the generated Ca²⁺ transients from w¹¹¹⁸; tinC-GCaMP2 before and after the administration of 10 uM diltiazem. F. Representative traces of the generated Ca²⁺ transients from w¹¹¹⁸; tinC-GCaMP2 before and after the administration of 2 uM thapsigargin. G–I. Summary data for the effects of thapsigargin on generated Ca²⁺ transients. The changes in the slopes of fluorescence increases (G), peak fluorescence (H), and slopes of fluorescence decreases (I) are shown. *p<0.05 by paired t-tests for changes in parameters before and after thapsigargin treatment (n=6).

Figure 5. The hdp²; tinC-GCaMP2 mutants have altered myocardial Ca²⁺ handling compared to w¹¹¹⁸; tinC-GCaMP2

A. Representative average pixel fluorescence intensity traces for w¹¹¹⁸; tinC-GCaMP2 (black) and hdp²; tinC-GCaMP2 (red) hearts. B–F. Measurements of ^dF/dtmax (panel B), the duration of the peak to 50% decay in fluorescence intensity (panel C), the duration of the 50% rise to peak in fluorescence intensity (panel D), FDHM (panel E), and the slope of linear decay from 80% to 20% fluorescence intensity (panel F) in w¹¹¹⁸; tinC-GCaMP2 (n=55 recordings from 19 individual preparations) and hdp²; tinC-GCaMP2 (n=81 recordings from 19 individual preparations) hearts. Individual measurements (black circles) and the mean (open circles) with SEM are shown. *p<0.05 for w¹¹¹⁸; tinC-GCaMP2 vs. hdp²; tinC-GCaMP2 for the indicated parameter.

Figure 6. Hearts from hdp² mutants have reductions in ryanodine receptor receptor transcript levels compared to w¹¹¹⁸

A. qPCR measurements of transcript expression levels in hearts from hdp² (open bars) relative to w¹¹¹⁸ (closed bars) for L-type Ca2+ channel (LTCC), ryanodine receptor(RYR) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), Sodium calcium exchanger (NCX) and inositol-3-phosphate receptor(IP3R). n=5 independent experiments with 60 fly hearts per group per experiment. *p<0.05 for hdp² vs. w¹¹¹⁸ by t-test. B. Representative traces of generated Ca²⁺ transients before and after the administration of 2 mM caffeine in w¹¹¹⁸; tinC-GCaMP2 hearts. C. Summary data for the fold changes in the area under the peaks of fluorescence after caffeine administration to w¹¹¹⁸; tinC-GCaMP2 vs. hdp²; tinC-GCaMP2 hearts.