Analysis of Drosophila cardiac hypertrophy by microcomputerized tomography for genetic dissection of heart growth mechanisms

Abstract

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here, we demonstrate that microcomputerized tomography (microCT) is an accessible, highly reproducible method for nondestructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85D^V12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca²⁺ entry (SOCE) driven by expression of constitutively active Stim (Stim^CA) or Orai (Orai^CA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca²⁺ influx channels. Intravital imaging of heart contractility revealed significantly reduced end-diastolic and end-systolic dimensions in Stim^CA- and Orai^CA-expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

NEW & NOTEWORTHY Genetic analysis of Drosophila cardiac hypertrophy holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. This potential has been hindered by a lack of rapid and effective methods for analyzing heart size in flies. Here, we demonstrate that analysis of the Drosophila heart with microcomputerized tomography yields accurate and highly reproducible heart size measurements that can be used to analyze heart growth and cardiac hypertrophy in Drosophila.

INTRODUCTION

Pathological cardiac hypertrophy occurs when the heart enlarges due to chronic functional overload. Although initially compensatory, unmitigated hypertrophy eventually transitions to heart failure (1–4). There are no cures for pathological cardiac hypertrophy or heart failure, and a better understanding of the cellular and molecular mechanisms that contribute to maladaptive cardiomyocyte growth is therefore essential.

Cardiac hypertrophy is driven by a complex integration of multiple signaling pathways including the Ca²⁺-calcineurin-NFAT, Ras-Raf-MAPK, and PI3K signaling axes (2). Resolution of this complexity will benefit from molecular pathway dissection in a genetically tractable model like Drosophila melanogaster. The Drosophila heart is a linear tube of cardiomyocytes that runs along the dorsal midline of the adult abdomen, and it pumps hemolymph throughout an open circulatory system (5–7). A major advantage of using Drosophila for cardiac research is the ability to rapidly engineer multiple genetic alterations in the same animal for integrated signaling pathway analysis. These approaches have shown that both Ras-Raf-MAPK and calcineurin-regulated signaling pathways drive cardiac hypertrophy in Drosophila (8–10), demonstrating that hypertrophic signaling in flies is well conserved with mammals. However, the full potential of Drosophila for cardiac hypertrophy research has been limited by a lack of readily accessible and reproducible methods for analyzing hypertrophic phenotypes in flies.

Key phenotypic indicators of cardiac hypertrophy include increased heart wall thickness and reduced luminal diastolic and systolic dimensions. Methods to analyze Drosophila heart contractile parameters including end-diastolic and end-systolic dimensions are well established (11). However, analyses of Drosophila heart wall thickness are limited and have relied on measurements derived from histopathological approaches involving physical sectioning of fixed and embedded flies (8–10, 12). Histopathology has several drawbacks that limit throughput, reproducibility, and accuracy. First, it is very time-consuming and laborious, and this limits the feasibility of processing large numbers of samples required for genetic pathway analyses. Second, achieving reproducible sectioning through the same region of the heart and in the same orientation is challenging. And third, physical sectioning through the heart yields relatively thick sections of 8–10 μm, which can limit the accuracy of individual measurements.

Several recent studies have described microcomputerized tomography (microCT) for quantitative analysis of internal organs in Drosophila (13–18), and our study aimed to determine the feasibility of microCT for quantitative analysis of cardiac hypertrophy in flies. MicroCT is an X-ray imaging methodology whereby the sample is incrementally rotated to generate a series of projection images that encompasses the full sample volume. Computerized algorithms then generate three-dimensional (3-D) tomogram reconstructions of the sample with isotropic resolution. Due to the small size of Drosophila, an entire animal at any developmental stage can be imaged in 3-D by microCT in a single acquisition sequence, allowing visualization of internal organs in any orientation and location with exceptional clarity and resolution (18). Importantly, microCT measurements can be reproducibly made from the same location within a specific organ or structure without imprecise physical sectioning. MicroCT also involves straightforward fixation and staining protocols, and multiple animals can be processed at a time. Reproducible measurements of wild-type Drosophila hearts have been made using microCT (19); however, whether microCT can be used to analyze pathological changes to heart architecture such as hypertrophy has not been determined. Integration of microCT with combinatorial Drosophila genetic tools has immense potential to provide new insights into molecular mechanisms of pathological heart remodeling.

Here, we demonstrate that analytical microCT approaches accurately and reproducibly report hypertrophy of the Drosophila heart. As proof of principle, we show using microCT that expression of constitutively active Ras, previously demonstrated to cause cardiac hypertrophy in Drosophila by histopathological analyses (8), results in significantly increased heart wall thickness in adult and larval flies. We then demonstrate using genetic approaches, microCT measurements, and contractility analysis that upregulation of store-operated Ca²⁺ entry (SOCE) drives cardiac hypertrophy in Drosophila. SOCE is a highly conserved Ca²⁺ signaling mechanism that couples influx of extracellular Ca²⁺ to depletion of endo/sarcoplasmic reticulum (E/SR) Ca²⁺ stores. SOCE is mediated by Ca²⁺-sensing Stim proteins in the E/SR and Orai Ca²⁺ influx channels in the plasma membrane (19–21). SOCE upregulation in mammalian cardiomyocytes is necessary and sufficient for pressure-overload-induced cardiac hypertrophy, likely due to SOCE activation of calcineurin signaling (22–24). However, mechanisms that regulate SOCE in cardiomyocytes, and how SOCE integrates with other hypertrophic signaling processes are poorly understood. Our results establish a new model of SOCE-driven cardiac hypertrophy that combines the power of Drosophila genetics with robust analysis of fly heart wall thickness with microCT. This microCT approach is straightforward, accessible, and readily adaptable to the renowned genetic tools in Drosophila, creating a powerful experimental platform for delineating the complex cellular and genetic processes that drive pathological cardiac hypertrophy.

MATERIALS AND METHODS

Fly Stocks

w1118 (RRID: BDSC_3605), UAS-Ras85D^V12 (RRID: BDSC_64195), and Orai RNAi (RRID: BDSC_53333) were from the Bloomington Drosophila Stock Center. tinC-GAL4 was from Dr. Manfred Frausch (Friedrich Alexander University). Flies expressing tdTomato under control of the cardiomyocyte-specific R94C02 enhancer (25) (CM-tdTom) were from Dr. Rolf Bodmer (Sanford Burnham Prebys Inst.). CM-tdTom² and CM-tdTom³ are second and third chromosome insertions, respectively. Wild-type UAS-Orai (Orai^WT) and constitutively active UAS-Orai (Orai^CA) flies were from Dr. Gaiti Hasan (National Center for Biological Sciences, Bangalore, India) (26). Flies that express wild-type Drosophila Stim (Stim^WT) under UASp control were generated by cloning the cDNA sequence of Drosophila Stim Isoform A from plasmid LD45776 (Drosophila Genomics Resource Center). Stim cDNA was inserted into vector pPWG (Carnegie Drosophila Gateway Vector Collection), which introduces a C-terminal EGFP tag and upstream UASp. For constitutively active Stim (Stim^CA), site-directed mutagenesis (Aglient QuikChange XL) was used to change aspartic acids 155 and 157 to alanines using StimWT as a template. Sequence confirmed plasmids were sent to BestGene for embryo injection. All UAS-Stim and Orai lines, including constitutively active mutants, were generated by random transgene integration in the same w1118 background. All of these stocks are homozygous viable, suggesting that transgene insertion did not disrupt essential genes.

Microcomputerized Tomography

The following microCT methods were adapted from Schoborg et al. (18, 27):

Adult labeling.

Adult female flies (7 day old; n = 5–20) were anesthetized with CO₂ and transferred to a 1.5-mL Eppendorf tube containing 1 mL of phosphate-buffered saline + 0.5% Triton-X 100 (0.5% PBST). Tubes were gently inverted and then incubated for 5 min at room temperature (RT) to remove wax cuticles. Flies were transferred to tubes containing 1 mL of Bouin’s fixative (5% acetic acid, 9% formaldehyde, 0.9% picric acid; Sigma) for 24 h. Samples were washed 3 × 30 min on a shaker in 1 mL microCT Wash Buffer (0.1 M Na₂HPO₄/NaH₂P0₄ + 1.8% sucrose, pH 7.0), followed by staining with 1 mL of 0.1 N iodine-iodide solution (Lugol’s solution) for 48 h. Flies were then washed twice with ultrapure water and stored at RT for up to 1 mo.

Larval labeling.

Third-instar larvae were placed in a 1.5-mL Eppendorf tube with 1 mL 0.5% PBST. Tubes were heated to 100°C for 20 s, then cooled to RT for 5 min. Larvae were then fixed in Bouin’s solution for 24 h and washed as for adults. Larval cuticles were then punctured at anterior and posterior ends with a microdissection needle to allow penetration of the labeling solution. Punctured larvae were incubated in Lugol’s solution for 48 h, washed twice with ultrapure water, and stored at RT for up to 1 mo.

Sample mounting and scanning.

Individual adults and larvae were placed head down in heat-sealed 10-µL micropipette tips containing ultrapure water, and a dulled 20-gauge needle was used to gently lower the animals until they fit snuggly in the taper of the pipette tip. Parafilm was wrapped around the base of the micropipette tip to prevent leakage. Samples were secured to the stage of the microCT scanner, pipette base down, using mounting putty. Samples were scanned with a Bruker SkyScan 1172 scanner controlled by Skyscan software operated on a Dell computer. The following X-ray source voltage and current settings were used: 40 kV, 110 μA, and 4 W. A Hamamatsu 10-Mp camera with 11.54-μm pixels coupled to a scintillator was used to collect X-rays and convert to photons. Fast scans were acquired with 2 × 2 binning and a source to sample distance of 44.4 mm to achieve an image pixel size on the detector of 2.85 μm. Slow scans did not use binning and had a source-to-sample distance of 28.7 mm, achieving an image pixel size of 1.15 μm. Slow scans also used 360° of sample rotation. Frame averaging was four for all scans. Importantly, the image pixel size does not fully reflect the actual image resolution of the reconstructed tomograms, as the actual spatial resolution also depends on other extrinsic factors including stain type, sample movement/deformation, scanner vibration, and reconstruction parameters. To empirically determine the actual spatial resolution of the SkyScan 1172 system, we measured line and point sizes generated by the QRM-MicroCT-BarPattern NANO (QRM GmbH, Mӧhrendorf Germany) when imaged using the imaging and reconstruction parameters described in Sample mounting and scanning and Reconstruction. These measurements indicated that the SkyScan 1172 produces tomograms with in-plane and axial spatial resolutions of 5–9 μm.

Reconstruction.

Tomograms were generated using NRecon software (Bruker, v1.7.0.4). The built-in shift correction function of NRecon, which uses reference scans to compensate for sample movement during scanning, was used for image alignment and reconstruction. Remaining misalignment was manually fine-tuned using the misalignment compensation function. Ring artifact correction was set to maximum 50 and beam hardening was 0%.

Heart wall thickness measurements.

Reconstructed microCT tomogram series were imported into FIJI (ImageJ v1.53c, NIH; RRID:SRC_002285) and viewed using the Orthogonal Views function to locate hearts in XY, XZ, and YZ orientations. Images that showed the largest opening of the conical chamber in cross section were used to measure the thickness of the heart walls on the two lateral sides, where the heart walls tended to be thickest and most easily isolated from other tissues or structures. For manual measurements, the boundaries of the heart walls were visually determined and line segments were manually drawn to directly measure the distance between the boundaries. For full-width at half-maximum (FWHM) measurements, lines were drawn that extended approximately 5–10 µm beyond the heart wall boundaries on either side. The “Plot Profile” function in FIJI was then used to generate a line scan of fluorescence intensity along the line, and the data were fit to a Gaussian function. FWHM was calculated using the formula FWHM = 2.355(d), where d is the standard deviation derived from the Gaussian fit. For both measurement methods, an average of 10 measurements from five slices was used to represent each animal. All measurements were made from randomized data files that were blinded to genotype.

Contractility Analysis

Intravital fluorescence imaging of hearts expressing CM-tdTom was carried out as previously described (28). Seven-day-old adult females were briefly anesthetized with CO₂ and adhered dorsal side down to glass coverslips with Norland Optical Adhesive cured with a 48-watt UV LED source (LKE) for 60 s. Animals recovered for 10 min before imaging. Hearts were imaged through the dorsal cuticle at 200 frames-per-second for 20 s using an ORCA-Flash4.0 V3 sCMOS camera (Hamamatsu) on a Nikon-Ti2 inverted microscope controlled with NIS-Elements software (RRID:SCR_014329). In addition, 550-nm excitation light was from a Spectra-X illuminator (Lumencor) and emission was collected through a 555–635-nm band-pass filter. To generate M-modes, a one-pixel-wide line was drawn through the heart in the A2 segment, and fluorescence intensity along this line was plotted using Multi-Kymograph in FIJI. The A2 segment was used in lieu of the conical chamber used for microCT measurements because the heart walls in the A2 segment are consistently aligned in parallel to one another, whereas the heart walls in the conical chamber are often aligned at irregular angles making consistent M-mode generation difficult. End-diastolic dimensions (EDD) and end-systolic dimensions (ESD) were calculated from the M-modes by measuring the distance between the heart walls at full relaxation and contraction, respectively. An average of five EDD and ESD measurements was calculated from each trace. Heart rate was calculated by counting the number of systoles over 20 s. FS was calculated as (EDD − ESD)/EDD × 100. All measurements were made from randomized data files that were blinded to genotype.

Statistical Analyses

Plots and statistical analyses were done with GraphPad Prism (RRID:SCR_002798). Contractility and heart wall measurements were analyzed by unpaired t test or one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance was at P < 0.05.

RESULTS

Quantitative microCT Analysis of Cardiac Hypertrophy in Adult Drosophila

We began validating our microCT methodology by analyzing iodine-labeled hearts in w1118 control adult flies crossed to the tinC-GAL4 driver used for heart-specific expression in subsequent experiments. We imaged adults by microCT at the highest resolution settings for which the entire animal fits within the detector’s field of view. These “slow scans” generate tomograms with 1.15-µm² pixels and an actual spatial resolution of 5–6 μm (See methods). Figure 1A shows a 3-D reconstruction of a wild-type w1118; tinC-GAL4 adult female fly generated from slow-scan microCT tomograms. The heart tube runs along the dorsal abdominal wall midline and is most easily identified in two-dimensional (2-D) views at the anterior and dorsal-most region of the abdomen (Fig. 1B). This anterior-most segment of the heart, which exhibits the largest luminal dimensions of the heart tube, is known as the conical chamber (Fig. 1, B–D). It can be identified in the XY view by locating the prominent dorsal longitudinal muscles (DLMs) of the thorax (Fig. 1C) and following them posteriorly to where they narrow and the abdomen begins. Because of the large luminal space of the conical chamber and the exceptional contrast and clarity provided by microCT, XY cross sections through the conical chamber are ideal for visualizing and measuring the heart walls (Fig. 1D). Note, however, that the lower or ventral heart walls are often obscured by the DLMs in XY cross sections through the conical chamber.

Figure 1.

MicroCT analysis of cardiac hypertrophy in adult Drosophila. A: 3-D reconstruction of a 7-day-old female fly generated from slow-scan microCT tomograms. XY, XZ, and YZ imaging planes are denoted by colored rectangles, and anatomical anterior (A), left (L), and dorsal (D) are indicated by white arrow coordinates. Yellow arrowheads indicate the location of the heart along the midline of the dorsal abdomen. B: slow-scan YZ-microCT tomogram at the midline of a 7-day-old w1118; tinC-GAL4 female fly, providing a longitudinal view of the animal. Head, thorax, and abdomen are indicated for orientation. The yellow dashed line outlines the heart (Ht) longitudinally from anterior to posterior of the dorsal abdomen. The thoracic dorsal longitudinal muscles (DLMs) are also indicated; note the region near the red arrow where the posterior DLMs are just beneath the anterior start of the heart, as this overlap is useful for identifying the heart conical chamber. C: single XY-tomogram from the location indicated by the green arrow in B, showing the anterior abdomen in cross section. The heart conical chamber in cross section (outlined in yellow dashes) is easily identified just dorsal to the small bundle of DLMs (outlined by red dashes). D: enlargements from B and C showing the heart conical chamber in longitudinal (yz; left) and cross section (XY; right) orientations. Yellow arrows denote the heart walls. E: longitudinal (YZ; left) and cross section (XY; right) images from slow-scan microCT tomograms of a tinC-GAL4>Ras85D^v12 7-day-old female fly. Yellow arrows denote the heart walls. Note the significant thickening of the heart walls compared with the w1118 control in D. F: representative cross-section images of heart conical chambers from fast-scan microCT tomograms of the same w1118; tinC-GAL4 and tinC-GAL4>Ras85D^v12 animals shown in B–E. Dashed yellow lines indicate the inner and outer boundaries of the heart walls. G: plot of manual heart wall thickness measurements from fast-scan microCT tomograms of w1118; tinC-GAL4 control and tinC-GAL4>Ras85D^v12 7-day-old females. Each circle represents measurements from a single animal (n = 22 for w1118; tinC-GAL4, 14 for tinC-GAL4>Ras85D^v12). H: plot of FWHM heart wall thickness measurements from fast-scan microCT tomograms of w1118; tinC-GAL4 control and tinC-GAL4>Ras85D^v12 7-day-old females. Each circle represents measurements from a single animal (n = 14 for w1118; tinC-GAL4, 14 for tinC-GAL4>Ras85D^v12). Red lines: median; black lines: quartiles. ****P < 0.0001, unpaired t test. Scale bars: B and C, 100 µm; D–F, 50 µm. FWHM, full-width at half-maximum; microCT, microcomputerized tomography; 3-D, three-dimensional.

We next determined whether microCT can be used to quantitatively analyze hypertrophy of the adult Drosophila heart by expressing a constitutively active Ras mutant (Ras85D^V12) previously shown to induce Drosophila cardiac hypertrophy (8). Heart-specific expression of Ras85D^V12 using tinC-GAL4 resulted in strikingly apparent thickening of heart walls in longitudinal and cross-section slow-scan images of the conical chamber compared with w1118; tinC-GAL4 controls (Fig. 1E, Supplemental Video S1; see https://doi.org/10.6084/m9.figshare.14976783, Supplemental Video S2; see https://doi.org/10.6084/m9.figshare.14976795). These slow scans provide maximal resolution for whole animal imaging; however, slow scans require 6–8 h to complete and are not practical for generating large datasets for quantitative analysis. To facilitate higher throughput for quantitative measurements, we next tested “fast scans” that require 20–30 min per animal and generate tomograms with 2.85 µm² pixels. Conical chamber cross sections from fast scans also clearly exhibited visual thickening of heart walls in Ras85D^V12 compared with w1118 control hearts (Fig. 1F). Direct measurement of heart wall thickness at the conical chambers from fast-scan, cross-section images from 22 w1118; tinC-GAL4 control animals yielded a mean of 7.97 ± 0.13 μm (Fig. 1G, means ± SE). Importantly, these measurements from microCT fast scans were nearly identical to previously published measurements from histopathological preparations of control hearts (8–10). Moreover, the low variance suggests a high degree of reproducibility of these microCT-based measurements. Measurements from 14 Ras85D^V12-expressing hearts revealed a significant, 33% increase in heart wall thickness compared with w1118; tinC-GAL4 (Fig. 1G). These results suggest significantly thicker heart walls for the Ras85D^V12-expressing animals. However, the difference between the means of the w1118 and Ras85D^V12 animals was 2.62 µm, slightly smaller than the camera pixel size for these fast scans. This raised the possibility that small differences in how the boundaries of the heart walls were visually determined could significantly impact the outcome of the measurements, though it should be noted that the low variance of our measurements suggests that this was not the case. To more rigorously test this possibility, we repeated our measurements by generating line scans of the fluorescence intensity across the heart walls and calculating the full-width at half maximum (FWHM) from Gaussian fits of the line scans. This method generates width measurements based on mathematical fit as opposed to manual determination of heart wall boundaries. The mean FWHM was 13.06 ± 0.19 µm for w1118; tinC-GAL4 animals and 17.74 ± 0.71 µm for Ras85D^V12-expressing animals (Fig. 1H). This was notably larger than the respective values from direct manual measurements, likely due to the influence of background fluorescence or fluorescence from closely adjacent structures on the Gaussian fit. Importantly, however, the difference between the means for the FWHM measurements, 35%, was nearly identical to the 33% difference for manual measurements, suggesting that both measurement methods report the same degree of hypertrophy of Ras85D^V12 hearts. We used direct manual measurements for subsequent analyses because we feel it provides a more accurate representation of the absolute width of the heart walls. Overall, these results demonstrate that our microCT methodology accurately reports the significant increase in heart wall thickness caused by Ras85D^V12 expression, suggesting that microCT imaging is a robust method for analyzing adult Drosophila cardiac hypertrophy.

MicroCT Analysis of Larval Heart Wall Thickness

Drosophila cardiomyocytes undergo significant growth during the larval developmental stages, making the larval heart useful for studying both physiological and pathological heart growth mechanisms (7, 29). As for adult flies, however, few tools are currently available for quantitative analysis of larval heart growth parameters such as heart wall thickness. We, therefore, assessed microCT for evaluating heart wall thickness in larvae. The heart spans the posterior third of third-instar larvae and is situated just under the dorsal cuticle (Fig. 2, A and B). In cross section, the heart appears as a round, thin-walled structure at the dorsal midline (Fig. 2C). It is important not to mistake the heart for the two tracheae, which are also round tubes found near the dorsal midline. The best way to identify the trachea is to find the two spiracles at the posterior end of the animal (indicated in Fig. 2B), and to then follow the spiracles into the animal where they become continuous with the trachea. The heart is then found between and just dorsal to the two tracheae (best seen in Fig. 2D). In fast scans, tinC-GAL4-driven Ras85D^V12 expression resulted in visible thickening of larval heart walls and narrowing of the heart lumen compared with w1118; tinC-GAL4 controls. (Fig. 2D). Direct measurements of heart wall thickness in cross section revealed a 26% increase in Ras85D^V12 compared with w1118; tinC-GAL4 hearts, from 7.24 ± 0.10 μm to 9.15 ± 0.26 µm, respectively (means ± SE; Fig. 2E). These results demonstrate that microCT generates images of Drosophila larvae with sufficient resolution and sensitivity to detect hypertrophy of the larval heart.

Figure 2.

MicroCT analysis of cardiac hypertrophy in Drosophila third-instar larvae. A: 3-D reconstruction of a third-instar larva generated from microCT tomograms. Anatomical anterior (A) and dorsal (D) are indicated by white arrow coordinates. Yellow arrowheads indicate the location of the heart along the dorsal midline in the posterior third of the animal. B: single slow-scan YZ-microCT tomogram at the midline of a w1118; tinC-GAL4 third-instar larva, providing a longitudinal view of the animal’s internal organs. Note that the larval anterior was not captured because the larva was larger than the detector’s field of view. The yellow dashed rectangle denotes the heart seen longitudinally, and the region within the rectangle is enlarged and contrast-enhanced in the lower right inset. Yellow arrowheads in the inset point to the heart walls. C: single XY-tomogram from the location indicated by the green arrow in B, showing the larva in cross section. The yellow dashed square denotes the heart, and the region within the square is enlarged and contrast-enhanced in the lower right inset. Yellow arrowheads in the inset point to the heart walls. D: representative cross-section images of control (w1118; tinC-GAL4) and tinC-GAL4>Ras85D^v12 third-instar larvae. Yellow squares denote the hearts as well as the areas enlarged in the bottom right insets. Dashed yellow lines in the insets indicate inner and outer boundaries of the heart walls. E: plot of heart wall thickness measurements from fast-scan microCT tomograms of control (w1118; tinC-GAL4) and tinC-GAL4>Ras85D^v12 third-instar larvae. Each circle represents measurements from a single animal (n = 14 for w1118; tinC-GAL4, 8 for tinC-GAL4>Ras85D^v12). Red lines: median; black lines: quartiles. ****P < 0.0001, unpaired t test. Scale bars = 100 µm. MicroCT, microcomputerized tomography; 3-D, three-dimensional.

SOCE Upregulation Results in Cardiac Hypertrophy

SOCE upregulation in cardiomyocytes is required for induction of pathological cardiac hypertrophy in vertebrates (22–24, 30, 31). Drosophila may be an important model to elucidate how SOCE regulates cardiac hypertrophy, but whether SOCE upregulation results in hypertrophy of the Drosophila heart is not known. We, therefore, used microCT to evaluate whether SOCE upregulation due to expression of constitutively active Stim and Orai mutants results in increased Drosophila heart wall thickness. Constitutively active Stim (Stim^CA) contains two aspartate to alanine substitutions within the EF-hand domain, causing Stim^CA to function in a Ca²⁺-unbound, fully active state (32). Constitutively active Orai (Orai^CA) has a glycine to methionine substitution in the channel hinge region, forcing the channel into an open conformation (33). Heart-specific Stim^CA and Orai^CA expression resulted in strikingly thicker heart walls in adults compared with controls when viewed in cross section (Fig. 3, A and B; Supplemental Video S3; see https://doi.org/10.6084/m9.figshare.14976828, Supplemental Video S4; see https://doi.org/10.6084/m9.figshare.14976834), similar to heart wall thickening seen with Ras85D^V12 (compare with Fig. 1F). The increase in heart wall thickness over controls in Stim^CA and Orai^CA animals was also quantitatively similar to that seen with Ras85D^V12 (43% and 39% for Stim^CA and Orai^CA, respectively; Fig. 3, C and D). Conversely, expression of wild-type Stim and Orai (Stim^WT and Orai^WT, respectively) did not result in significant changes to heart wall thickness compared with controls (Fig. 3, A–D). Stim^CA expression also resulted in significantly increased heart wall thickness in third-instar larvae (Supplemental Fig. S1; see https://doi.org/10.6084/m9.figshare.14976753), suggesting that upregulated SOCE can drive excessive cardiomyocyte growth in the developing heart. These results demonstrate that as in mammals, SOCE upregulation results in hypertrophy of the Drosophila heart, and that microCT is a valuable tool when combined with genetic approaches for analysis of hypertrophic signaling.

Figure 3.

SOCE upregulation causes Drosophila cardiac hypertrophy. A: representative images from microCT fast scans showing heart conical chambers in cross section from control (w1118; tinC-GAL4) and tinC-GAL4-driven Stim^WT and Stim^CA-expressing 7-day-old females. Dashed yellow lines indicate the inner and outer boundaries of the heart walls. B: representative images from microCT fast-scan images showing heart conical chambers in cross section from control (CM-tdTom²; tinC-GAL4) and tinC-GAL4 driven Orai^WT and Orai^CA-expressing 7-day-old females (these animals had CM-tdTom² because the same cross was also used for intravital imaging). Dashed yellow lines indicate inner and outer boundaries of the heart walls. C and D: plots of heart wall thickness measurements from fast-scan microCT tomograms of 7-day-old females with genotypes shown in A and B, respectively. Each circle represents measurements from a single animal (n = 11 for w1118; tinC-GAL4; 10 for Stim^WT, 12 for Stim^CA, 7 for CM-tdTom²; tinC-GAL4, 7 for Orai^WT, and 8 for Orai^CA). Red lines: median; black lines: quartiles. ns, not significant, ****P < 0.0001 compared with control, one-way ANOVA with Tukey’s multiple comparisons. Scale bars = 25 µm. Anatomical anterior (A) and dorsal (D) are indicated by white arrow coordinates. MicroCT, microcomputerized tomography; SOCE, store-operated Ca²⁺ entry.

Altered cardiac dimensions during the contractile cycle are another key feature of cardiac hypertrophy. Contractile alterations characteristic of hypertrophy include significantly decreased end-diastolic and end-systolic dimensions without changes to fractional shortening. These diastolic and systolic alterations can be caused by multiple factors including cardiomyocyte growth, fibrosis, and altered Ca²⁺ handling (34). We, therefore, determined next whether SOCE upregulation impairs systolic and diastolic dimensions of adult Drosophila hearts. Heart contractions were analyzed by intravital fluorescence imaging of animals with cardiomyocyte-specific tdTomato (CM-tdTom) expression (25, 28). Control hearts exhibited a mean end-diastolic dimension (EDD) of 65.17 ± 1.33 μm, mean end-systolic dimension (ESD) of 32.15 ± 0.83 μm, and mean fractional shortening (FS) of 51 ± 0.85% (means ± SE; Fig. 4, A–D). These parameters were not significantly altered by expression of Stim^WT or Orai^WT, consistent with the lack of cardiac hypertrophy shown by microCT analysis. In striking contrast, EDDs were significantly reduced compared with controls by 26%, 32%, and 15% for Stim^CA, Orai^CA, and Ras85D^V12, respectively (Fig. 4, A and B). ESDs were also significantly reduced in Stim^CA, Orai^CA, and Ras85D^V12-expressing hearts (Fig. 4C), whereas FS was unaltered with Stim^CA or Orai^CA and modestly increased with Ras85D^V12 (Fig. 4D). Heart rates were similar across control and experimental groups (Fig. 4E). These results demonstrate that SOCE upregulation results in heart remodeling that reduces end-diastolic and end-systolic dimensions, consistent with the hypertrophic phenotype observed in microCT analyses.

Figure 4.

SOCE upregulation impairs heart contractility. A. heart contractility was analyzed by intravital fluorescence imaging of 7-day-old females expressing CM-tdTom. Shown are representative M-modes from animals with tinC-GAL4 and CM-tdTom² (control), and tinC-GAL4-driven Stim^WT, Orai^WT, Stim^CA, Orai^CA, and Ras85D^V12. The red line in the CM-tdTom² trace depicts systole and the yellow line depicts diastole. Plots of EDD (B), ESD (C), FS (D), and HR (E) calculated from M-modes of the genotypes are shown in A. Each circle represents measurements from a single animal (n = 37 for w1118, 24 for Stim^WT, 15 for Stim^CA, 12 for Orai^WT, 12 for Orai^CA, and 20 for Ras85D^V12). Red lines: median; black lines: quartiles. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with control, one-way ANOVA with Tukey’s multiple comparisons. ns, not significant; SOCE, store-operated Ca²⁺ entry.

Stim^CA-Mediated Cardiac Hypertrophy Requires Orai Channels

As final proof of principle for how microCT can be combined with powerful genetic tools to understand heart pathophysiology, we used a genetic suppressor approach to determine whether Stim^CA-induced cardiac hypertrophy requires Orai channels. MicroCT analysis again showed significant heart wall thickening in animals with Stim^CA expression alone (Fig. 5, A and C). Remarkably, however, the hypertrophic phenotype was completely suppressed by coexpression of Stim^CA with an Orai RNAi construct that we previously demonstrated resulted in ∼80% suppression of Orai mRNA expression (28), as heart wall thickness in these animals was similar to controls, as well as to animals with Orai RNAi alone (Fig. 5, A and C). This strongly suggests that hypertrophy of Stim^CA-expressing hearts results from upregulated Ca²⁺ influx through Orai channels. In further support of this conclusion, hypertrophy caused by Ras85D^V12 expression was not similarly suppressed by coexpression of Orai RNAi (Fig. 5, B and D). Thus, the ability of Orai knockdown to suppress hypertrophy is specific to SOCE upregulation, as opposed to a generalized ability of Orai suppression to universally repress hypertrophic growth. This Ras85D^V12 result also suggests that coexpression of transgenes with Orai RNAi does not significantly reduce transgene expression, though we cannot be sure how much Ras85D^V12 versus Stim^CA is required to drive hypertrophic cardiomyocyte growth. We did not directly determine amounts of Ras85D^V12 or Stim^CA coexpression with Orai RNAi by Western blot or qRT-PCR because of challenges inherent to isolating sufficient, homogeneous quantities of Drosophila cardiomyocytes. Finally, we determined whether the deleterious effects of Stim^CA expression on contractile heart dimensions similarly depend on Orai channels. In support of this, coexpression of Stim^CA with Orai RNAi reversed the reductions in EDD and ESD seen with Stim^CA expression alone (Fig. 6, A and C–E). Notably, Orai RNAi expression alone resulted in significant increases in EDD and ESD compared with controls (Fig. 6, A and C–E), consistent with dilated cardiomyopathy that results from SOCE suppression (28). The effect of Orai suppression was again specific to Stim^CA, as reductions in EDD and ESD caused by Ras85D^V12 expression were not similarly reversed by coexpression with Orai RNAi (Fig. 6, B, F–H). Collectively, these results demonstrate that upregulated SOCE signaling mediated by both Stim and Orai is sufficient to drive cardiac hypertrophy in Drosophila. We further show that the effects of SOCE upregulation are specific and distinct from other mechanisms that drive hypertrophic growth of the heart such as upregulated Ras signaling.

Figure 5.

Stim^CA-driven hypertrophy requires Orai channels. Representative microCT fast-scan images showing heart conical chambers in cross section from control (CM-tdTom²; tinC-GAL4) and tinC-GAL4-driven Stim^CA, Stim^CA + Orai RNAi, and Orai RNAi-expressing 7-day-old females (A), and control (CM-tdTom³; tinC-GAL4) and tinC-GAL4-driven Ras85D^v12, Ras85D^v12 + Orai RNAi, and Orai RNAi-expressing 7-day-old females (B) (these animals had CM-tdTom because the same cross was also used for intravital imaging). Dashed yellow lines indicate inner and outer boundaries of the heart walls. C and D: plots of heart wall thickness measurements from fast-scan microCT tomograms of 7-day-old females with the genotypes shown in A and B, respectively. Each circle represents measurements from a single animal (n = 7 for CM-tdTom², 8 for Stim^CA, 8 for Orai RNAi + Stim^CA, 7 for Orai RNAi, 7 for CM-tdTom³, 7 for Ras85D^V12, 8 for Ras85D^V12 + Orai RNAi, 7 for Orai RNAi). Red lines: median; black lines: quartiles. **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons. Scale bars = 25 µm. Anatomical anterior (A) and dorsal (D) are indicated by white arrow coordinates. microCT, microcomputerized tomography; ns, not significant.

Figure 6.

Impaired heart contractility caused by Stim^CA expression requires Orai channels. Representative M-mode traces from 7-day old female flies with tinC-GAL4 + CM-tdTom² (control) and tinC-GAL4 driven Stim^CA, Orai RNAi + Stim^CA, and Orai RNAi (A), and tinC-GAL4 + CM-tdTom3 (control) and tinC-GAL4 driven Ras85D^V12, Ras85D^V12 + Orai RNAi, and Orai RNAi (B). Plots of EDD (C), ESD (D), and FS (E), calculated from M-mode traces of the genotypes shown in A. Plots of EDD (F), ESD (G), and FS (H), calculated from M-mode traces of the genotypes shown in B. Each circle represents measurements from a single animal (n = 17 for CM-tdTom², 11 for Stim^CA, 12 for Stim^CA + Orai RNAi, 16 for Orai RNAi, 19 for CM-tdTom³, 14 for Ras85D^V12, 15 for Ras85D^V12 + Orai RNAi, 15 for Orai RNAi). Red lines: median; black lines: quartiles. ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test.

DISCUSSION

We have demonstrated that microCT is a powerful, nondestructive imaging platform for analysis of Drosophila heart architecture that when combined with genetic tools and functional heart analysis, can be used to decipher complex molecular mechanisms of heart growth and pathological remodeling. Our introduction of microCT imaging to the Drosophila heart analysis toolkit has the potential to significantly accelerate our understanding of conserved genetic and signaling mechanisms that regulate heart growth during development and pathological transformations that lead to hypertrophy and heart failure. In contrast to histopathological approaches previously used to analyze Drosophila cardiac hypertrophy, we show that microCT yields heart size measurements in flies that are rapidly obtained, highly reproducible, and sensitive enough to detect changes in heart wall thickness indicative of hypertrophy. And importantly, microCT tomograms can be used to assemble complete 3-D reconstructions of whole animals, such that the entire heart and other organs can be visualized and measured isometrically in any orientation. Thus, multiple landmarks external to the heart can be used to position consistent measurements, and measurements can always be made from images with the same anatomical orientation. The ability to analyze multiple organs in the same animal with one single data set also means that microCT offers nearly unmatched potential for studying multisystem diseases that affect the heart as well as other organs (35). From a practical standpoint, microCT instruments are commonly found in preclinical, small animal imaging facilities at research institutes, and may already be accessible to many Drosophila research laboratories. Alternatively, the cost of new microCT instruments is similar to that of low-end confocal microscope systems, though microCT instruments vary widely with respect to cost and functionality.

We validated our microCT approach for detecting Drosophila cardiac hypertrophy by analyzing animals that express constitutively active Ras, an established hypertrophic model based on histopathological analysis (8). We used female animals throughout our experiments because female adults are larger than males and facilitate easier, more accurate measurements of heart architecture, though it should be noted that males are also suitable for microCT analysis (18, 27). Our microCT heart wall thickness measurements in adult controls were consistent with measurements from histopathological preparations, indicating a normal heart wall thickness of 6–8 μm in the conical chamber. Ras85D^V12 expression resulted in a 3 to 4 μm, or ∼30%–50% increase compared with controls by our microCT measurements, indicating significant hypertrophy of the heart walls. Notably, however, prior histopathological measurements showed a ∼15-μm increase in thickness with Ras85D^V12 expression (8). A possible reason for this apparent discrepancy is that histopathological measurements were made from 8-μm-thick heart sections, whereas our microCT measurements were from 2.85-μm optical slices. The increased thickness of histopathological tissue sections may have contributed to the thicker appearance of the heart walls, especially if the hearts were not oriented perpendicular to the plane of section, or if there were heart wall deformations within the section. Thus, we expect that the thinner sections used with microCT result in more accurate measurements of heart wall thickness compared with histopathology.

Heart wall measurements derived from microCT tomograms can be influenced by multiple experimental and technical factors that should be considered when analyzing experimental outcomes. First, the effective resolution of the microCT system will impact size measurements as well as the ability to visualize closely spaced objects or structures. Our heart wall thickness measurements consistently yielded values in control animals ranging from 6 to 8 µm, which is close to the resolution limit of 5–9 µm for the fast-scan tomograms used. We therefore cannot rule out the possibility that heart walls are somewhat thinner than reported in control animals. This also indicates that our microCT system would not be appropriate for analysis of heart wall thinning, such as in dilated cardiomyopathy, or for analysis of single cardiomyocytes or subcellular structures. However, our results clearly demonstrate that we can effectively detect increases in heart wall thickness on the order of 25%–35%, indicative of hypertrophy, though we cannot determine at this time the lower limit of detectable hypertrophy with our methods. Our ability to detect increases in heart wall thickness is also consistent with a previous report showing that small changes in heart wall width along the length of a single heart can be detected by microCT imaging with a resolution similar to ours (18). This ability to detect hypertrophy is possible because the overall thickness of the hypertrophied hearts of 10–12 µm is well above the resolution limit, and imaging was done with two to three times oversampling due to 2.85-µm² pixels. Another important consideration specific to heart analysis by microCT is the contractile state of the heart at the time of fixation and imaging. Fixation of hearts at different stages of the contractile cycle would likely result in different heart wall thicknesses because individual cardiomyocytes shorten and become thicker when the heart contracts. Our protocol initiates fixation after flies have been submerged in PBST for 5 min, such that the flies are likely dead and their hearts no longer contracting at the time of fixation. It is likely then that the hearts are consistently fixed in a relaxed, diastolic state, and the low variance of our heart wall measurements would support this sample-to-sample consistency. To more rigorously test this, we measured ESD and EDD of CM-tdTom-expressing hearts by intravital imaging before and immediately following soaking the animals in PBST for 5 min, when fixation for microCT preparation would occur. As expected, we found that hearts had completely stopped beating after 5 min in PBST, and the dimensions of the stopped hearts closely matched EDDs measured before PBST treatment (Supplemental Fig. S2; see https://doi.org/10.6084/m9.figshare.17026631). Thus, we can conclude that our microCT measurements are consistently made on hearts that are in a fully relaxed, diastolic state.

In addition to heart wall measurements, analysis of cardiac dimensions during contractility is also important to defining cardiac hypertrophy phenotypes. MicroCT requires fixation and is therefore not amenable to analysis of contracting hearts in live animals. We, therefore, combined our microCT approaches with intravital imaging of heart contractility. Our analyses showed significantly reduced EDDs and ESDs in Ras85D^V12, Stim^CA, and Orai^CA-expressing hearts, consistent with thickened heart walls. Because both EDD and ESD were nearly equally affected in hypertrophic hearts compared with controls, FS was not significantly altered indicating similar overall displacement of the heart walls. It is noteworthy that in microCT images of these hypertrophic hearts, the luminal dimensions of the hearts were significantly reduced compared with controls. This likely reflects the reduced diastolic dimensions measured from contractility analyses. Direct measurement of luminal diameter or area from microCT images could therefore serve as an additional analytical approach to defining hypertrophy though we did not attempt these measurements in our current study.

We also demonstrated the utility of microCT for analysis of the larval Drosophila heart. This is significant because substantial physiological heart growth in Drosophila occurs during the larval developmental stages (5, 7, 11). Thus, microCT may be a valuable tool for delineating physiological mechanisms of developmental heart growth. And because many key signaling mechanisms that drive developmental heart growth are reactivated during pathological cardiac hypertrophy and heart failure, microCT analysis of the larval heart may also be beneficial for uncovering new mechanisms involved in heart failure (36, 37).

To demonstrate the power of microCT for analysis of signaling pathways that drive cardiac hypertrophy, we used this technique to investigate the role of SOCE signaling in hypertrophy of the Drosophila heart. Upregulated SOCE is essential for induction of pathological cardiac hypertrophy (23, 24, 30, 38, 39). However, we still have much to learn about the precise roles of SOCE signaling in hypertrophic cardiomyocyte growth. Furthermore, a growing number of gain-of-function STIM1 and Orai1 mutations have been identified in human patients (40). Whether these gain-of-function mutations affect human heart physiology is unclear, further highlighting significant gaps in our understanding of SOCE function in the heart. Genetic models of upregulated SOCE are therefore vital to understanding the mechanisms by which SOCE regulates cardiac physiology and disease pathogenesis. Our microCT and contractility data show that genetic SOCE upregulation in cardiomyocytes by Stim^CA and Orai^CA expression results in significant hypertrophy of the Drosophila heart, similar to Ras85D^V12. The effects of SOCE upregulation on heart function were pathological, as the number of heart-specific Stim^CA-expressing animals reaching the pupal stage of development was reduced by ∼70% compared with controls, and only ∼20% of Stim^CA animals reached adulthood (Supplemental Fig. S3; see https://doi.org/10.6084/m9.figshare.14976765). We also found that Stim^CA-expressing adults were difficult to maintain, suggesting reduced adult viability (data not shown). It should be noted that our experiments with Stim^CA and Orai^CA were done with single transgene integration lines for each construct, and we cannot rule out the possibility that changes in heart size and animal physiology were the result of genomic disruptions due to transgene integration alone. Our results may also depend on the temporal and spatial characteristics of transgene expression driven by tinC-GAL4. For example, tinC-GAL4 is known to drive the highest expression in the pairs of adult cardiomyocytes that comprise the ostia, and expression may also be higher overall in the larval compared with adult heart (41).

The similar results observed for both Stim^CA and Orai^CA expression are consistent with a mechanism whereby Ca²⁺ influx through Stim-activated Orai channels is sufficient for induction of cardiac hypertrophy. However, it has been suggested that Stim regulates Orai-independent targets in cardiomyocytes including l-type Ca²⁺ channels and phospholamban (42, 43). We found that hypertrophy of Stim^CA-expressing hearts was suppressed by coexpression of Orai RNAi, suggesting that Stim^CA-induced hypertrophy specifically requires Orai channels as opposed to Orai-independent targets in Drosophila. In contrast to Stim^CA, Ras85D^V12-induced hypertrophy was not suppressed by Orai RNAi, suggesting that Orai suppression does not have a generalized antihypertrophic effect and is specific to hypertrophy caused by Stim activation. This result also suggests that Ras and SOCE function in parallel hypertrophic signaling pathways, as opposed to a linear pathway in which Ras functions upstream of SOCE.

Accumulating evidence suggests that SOCE acts through calcineurin signaling to drive hypertrophic cardiomyocyte growth (22, 23, 30, 38). However, additional targets of upregulated SOCE in cardiomyocytes have also been reported including CaMKII (23) and mTORC2/Akt (38). Thus, consistent with the complex and multifaceted etiology of pathological cardiac hypertrophy, the role of SOCE in this disease likely involves multiple targets and regulatory processes. Addition of microCT to the Drosophila heart analysis toolkit will significantly advance our ability to genetically dissect these complex signaling mechanisms.

SUPPLEMENTAL DATA

Supplemental Video S1: https://doi.org/10.6084/m9.figshare.14976783

Supplemental Video S2: https://doi.org/10.6084/m9.figshare.14976795

Supplemental Video S3: https://doi.org/10.6084/m9.figshare.14976828

Supplemental Video S4: https://doi.org/10.6084/m9.figshare.14976834

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.14976753

Supplemental Fig. S2: https://doi.org/10.6084/m9.figshare.17026631

Supplemental Fig. S3: https://doi.org/10.6084/m9.figshare.14976765.

GRANTS

J.T.S. was supported by funding from the Collaborative Health Initiative Research Program (CHIRP) of the National Institutes of Health (NIH), from NIH Grant 1 R21 NS121821-01, and from Uniformed Services University Grant I 80 VP000003. T.A.S. was supported by NIH Grant 1 K22 HL137902-01 and an Institutional Development Award (IDeA) from NIH Grant 2 P20 GM103432. Reagents obtained from the Bloomington Drosophila Stock Center (NIH Grant P40OD018537) and Drosophila Genomics Resource Center (NIH Grant 2P40OD010949) were used in this study.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.E.P. and J.T.S. conceived and designed research; C.E.P. and B.A.T. performed experiments; C.E.P. and T.A.S. analyzed data; C.E.P., T.A.S., and J.T.S. interpreted results of experiments; C.E.P. and J.T.S. prepared figures; C.E.P. drafted manuscript; T.A.S. and J.T.S. edited and revised manuscript; C.E.P., B.A.T., T.A.S., and J.T.S. approved final version of manuscript.