Author response: Age-dependent diastolic heart failure in an in vivo Drosophila model (2017)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract While the signals and complexes that coordinate the heartbeat are well established, how the heart maintains its electromechanical rhythm over a lifetime remains an open question with significant implications to human health. Reasoning that this homeostatic challenge confronts all pulsatile organs, we developed a high resolution imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. We demonstrate that, as in humans, normal aging primarily manifests as defects in relaxation (diastole) while preserving contractile performance. Using this approach, we discovered that a pair of two-pore potassium channel (K2P) subunits, largely dispensable early in life, are necessary for terminating contraction (systole) in aged animals, where their loss culminates in fibrillatory cardiac arrest. As the pumping function of its heart is acutely dispensable for survival, Drosophila represents a uniquely accessible model for understanding the signaling networks maintaining cardiac performance during normal aging. https://doi.org/10.7554/eLife.20851.001 Introduction During an average human life span, the heart will undergo 2.8 billion cycles of contraction and relaxation, wherein its four chambers rhythmically beat in a precisely choreographed sequence to efficiently circulate the blood. However, despite our understanding of the molecular basis of the heartbeat (Bers, 2008; Monfredi et al., 2013; Solaro, 2010), societies must confront an increasing failure of this process, which represents an intersection between normal aging and deleterious environmental and genetic factors (Yancy et al., 2013). Previous research has demonstrated that many biophysical properties of the heart are altered over the course of a lifetime (Carrick-Ranson et al., 2012; Cheng et al., 2009; Lakatta et al., 2014). These changes exist structurally, with myocardial fibrosis and arterial stiffening playing a leading role, but also manifest functionally, with alterations in the electrical waveform and calcium handling. However, whether these changes reflect normal senescence or compensatory attempts to maintain function remains an intense area of investigation. The maintenance of cellular and organismal physiology, termed homeostasis, is essential for all living organisms. To uncover the mechanisms that maintain cardiac function, it has been necessary to develop a systems perspective (Kohl et al., 2010). These efforts have revealed that the heart's rhythmic behavior is not uniform but exhibits variations that precisely match local mechanical needs (Brutsaert, 1987; Schram et al., 2002). Alterations in the heart's rhythm also occur during normal aging, where the action potential is prolonged in an apparent effort to maintain the intracellular calcium dynamics necessary for contraction (Janczewski et al., 2002). Such electrical remodeling is also observed in chronic heart failure, atrial fibrillation and in several genetic and pharmacological models, suggesting that ion channels are under homeostatic regulatory control (Nattel et al., 2007; Rosati and McKinnon, 2004; Schmitt et al., 2014). These observations raise the possibility that the heart monitors its own efficacy and adaptively remodels its electromechanical tone. However, significant implications to the pathophysiology of heart disease notwithstanding, the extended lifetimes and complexity of traditional mammalian models, as well as the necessity of their cardiovascular system for organismal survival, have confounded our ability to examine these potential homeostatic responses. In seeking to develop a more tractable model for understanding this process, we reasoned that all electromechanical biological oscillators must defend themselves against intrinsic and environmental instability and compensate for natural senescence if they are to maintain their efficacy over time. The first primordial cardiomyocytes likely originated in Bilateria, and the transcription factors tinman (Nkx2.5 in mammals), hand and mef2 represent a conserved cardiogenic program linking the evolution of Protostome and Deuterostome hearts (Bishopric, 2005). Despite having an open circulatory system that reverses the direction of flow periodically (Wasserthal, 2007) and a greatly simplified architecture (Rotstein and Paululat, 2016), Drosophila has proven to be of significant utility in the study of cardiac development and physiology, revealing mechanisms of cardiogenesis and heart function that are highly conserved with mammals (den Hoed et al., 2013; Frasch, 2016; Monnier et al., 2012; Neely et al., 2010). Consequently, Drosophila serves as a uniquely high-throughput 'pioneer' genetic model for uncovering conserved pathways involved in cardiomyocyte development and function. A variety of imaging modalities have investigated the molecular mechanisms underlying cardiac function in Drosophila (Ocorr et al., 2014). Most notably, an in situ preparation has been used to isolate the intrinsic regulators of cardiac performance in a defined physiological solution and in the absence of neuronal input. A complementary approach would be to monitor heart function in intact animals, where the full suite of intrinsic, environmental and homeostatic processes regulating the heart would be accessible to investigation, with minimal decay in cardiac performance for hours after preparation. While simple transmitted or reflected luminance measures can robustly monitor the rhythm of the heart in vivo, one cannot accurately measure heart wall displacement using this approach. Optical coherence tomography has been utilized to measure heart rhythm and displacement in vivo, and has successfully uncovered several novel genes affecting cardiac function (Choma et al., 2006; Wolf et al., 2006; Li et al., 2013; Alex et al., 2015). However, increasing heart wall contrast relative to the lumen and surrounding tissues could yield further improvements in our ability to assess cardiac function in the intact animal. In this study, we present a high resolution fluorescence imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. Using this platform, we demonstrate that, as in humans, normal aging manifests primarily as defects in relaxation (diastole) while preserving contractile performance, suggesting a conserved susceptibility to aging-related declines in the electrical, biochemical or structural processes facilitating relaxation. We also uncover a critical role for a heteromeric two-pore potassium channel in maintaining cardiac rhythmicity during aging, which appears dispensable for heart function early in life but is critical for preventing fibrillatory cardiac arrest in aged animals. We propose that the robustness, speed and resolution of this in vivo platform will significantly increase the utility of Drosophila in understanding conserved mechanisms of cardiac aging and homeostasis. Results Imaging cardiac performance in intact, unanesthetized Drosophila We developed a fluorescence-based approach for imaging the heart in intact, unanesthetized Drosophila. Briefly, the red fluorophore tdTomato was transgenically expressed in working cardiomyocytes using a newly discovered heart enhancer R94C02 (Tables 1 and 2). Intact flies were attached to coverslips using ultraviolet-activated optical cement and placed in the optical path (Figure 1A). Excitation light was spatially limited to the region of the dorsal abdomen containing the second and third chambers of the heart using a digital-micromirror projector, with the fluorescence emitting back through the cuticle captured on a sCMOS camera operating at 120 frames per second (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset Imaging cardiac performance in intact, unanesthetized Drosophila. (A) Full view of the intact preparation, with imaging region of interest (blue). The head and legs are freely moving, while the wings, dorsal thorax and dorsal abdomen are affixed to the coverglass with optical cement. (B) Electro-optical diagram of the imaging system. (C) Single anterograde heartbeat at half frame-rate with heart wall position (yellow), initiation of contraction (red triangles) and of relaxation (blue triangles) calls. (D) Associated YT kymograph (magenta) with heart wall detection (white dots). (E) Corresponding digitization, segmented into anterograde (white) and retrograde (grey) heartbeat epochs. The triangles denote the initiation (red) and end (blue) of contractions. (F) Two-dimensional probability map of heart chamber diameter and heartbeat duration with median +/− quartile overlay for systole (red) and diastole (blue). (G) Two-dimensional probability map of fractional shortening and systolic interval. All data in this figure are representative and from the 10-day-old w1118 wild-type dataset. See Materials and methods for all functional parameter definitions and their derivation. Scale bars: (black vertical) 75 µm, (blue horizontal) 1 s. See also Video 1. https://doi.org/10.7554/eLife.20851.002 Using this in vivo preparation and an automated feature detection algorithm based on maximal contrast, we unambiguously tracked the heart wall across the cardiac cycle in intact animals (Figure 1C,D). We then developed a segmentation algorithm that converted this digital representation of chamber diameter over time into discrete contraction and relaxation events (Figure 1C–E, Video 1). This segmentation allowed us to derive a diverse set of heart functional parameters, including estimates of cardiac output and stroke volume. We refer the reader to the Materials and methods section for a detailed explanation of the algorithms and formulas used. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Heartbeat visualization, digitization and segmentation. One-third speed video of the 10 day adult female displayed in Figure 1C–E, with heart wall position calls (yellow) and the attending transformation into heart chamber diameter as a function of time in a 1 s streaming window. The initiation and end of each contraction are specified by a red and blue triangle, respectively. Note the periodic reversal in the direction of heartbeat peristalsis. https://doi.org/10.7554/eLife.20851.004 In wild-type animals, heartbeats are very consistent and can be visualized en masse by assembling a two-dimensional probability map of chamber diameter across the cardiac cycle (Figure 1F) or fractional shortening versus the systolic interval (Figure 1G), which measures the percentage reduction in chamber diameter during a contraction. Both visualizations demonstrated that the mechanical rhythm of the heart is reproducible between animals. This consistency is also evident in the four primary measurements of the cardiac cycle: the systolic interval, diastolic interval, end systolic diameter, and end diastolic diameter. The standard deviations for these four measurements are between 7% and 11% for our wild-type dataset of young animals between 10 and 30 days of age (n.s., Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparisons test, n = 68), demonstrating that this in vivo preparation is highly reproducible. We next performed a series of control experiments to assess the stability of the preparation. The excitation light intensity utilized did not appreciably alter the heartbeat waveform but at higher light intensities, the kinetics of contraction and relaxation accelerated significantly (Figure 1—figure supplement 1A,B, R2 = 0.93). Furthermore, the preparation was stable for at least two hours, with cardiac output maintained even after 19 hr in flies kept hydrated overnight (Figure 1—figure supplement 1C,D), establishing that our approach is not subject to meaningful variability associated with decay in the health of the preparation. Normal aging manifests as diastolic dysfunction while preserving contractile performance While modifiable risk factors including elevated blood pressure, tobacco use, abnormal blood sugar levels, physical inactivity and obesity strongly exacerbate the incidence of heart disease (Yancy et al., 2013), aging-related changes in the structure and physiology of the heart also influence disease progression (Lakatta et al., 2014). Increases in arterial load, ventricular hypertrophy and diastolic dysfunction are well established in healthy aging hearts (Carrick-Ranson et al., 2012; Cheng et al., 2009; Lakatta et al., 2014). At the molecular level, animal models have revealed clear alterations in action potential duration and calcium handling with age (Feridooni et al., 2015). However, the relative contribution of physiological remodeling in cardiomyocytes to declines in cardiac performance has proven difficult to deconvolve from defects arising from arterial overload and structural hypertrophy. To establish whether our Drosophila platform might represent a simplified model to study cardiomyocyte senescence in vivo, we aged female flies to determine how heart function would change over time. Previous research using an in situ dissected preparation in Drosophila has demonstrated progressive declines in cardiac rhythmicity during the first five weeks of life (Ocorr et al., 2007). In our in vivo preparation, the heart rate and cardiac performance of flies was remarkably stable for up to 30 days of age in two 'wild-type' genetic backgrounds, w1118 and Canton S, which we subsequently grouped for further analysis (6.9% maximal variance in heart rate between these two groups at 10, 20 and 30 days of age, n.s., Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparisons test). At 50 days of age, flies displayed a small decrease in heart rate, (Figure 2A, R2 = 0.84) which primarily reflected a prolongation of the systolic interval (Figure 2—figure supplement 1A). Only after 50 days of age did the diastolic interval lengthen (Figure 2—figure supplement 1B) and cardiac output per second decline (Figure 2B). Figure 2 with 1 supplement see all Download asset Open asset Normal aging is characterized by a diastolic decline with preserved contractility. Various cardiac functional parameters presented by age in a combined w1118 and Canton S dataset, n = 18 to 30 animals per time-point: (A) Median heartbeat with heart rate (inset). (B) Estimated cardiac output per second (blue) and per stroke (red). (C) Median heart wall velocity with peak velocities of contraction (red dots) and relaxation (blue dots). (D) Probability histograms of the time from initiation of contraction to peak contraction velocity (red) and from the peak contraction velocity to peak relaxation velocity (blue). The shaded areas in panel B represent the mean +/− s.d., with regressions plotted as dotted lines. https://doi.org/10.7554/eLife.20851.005 Figure 2—source data 1 Median heartbeats for all individual animals in panel A. Median heartbeats were calculated for individual animals (Table) and for all consolidated heartbeats for a respective age (Panel A and last column of each Table). These source data provide a representation of the observed heartbeat waveform variability between animals. https://doi.org/10.7554/eLife.20851.006 Download elife-20851-fig2-data1-v1.xlsx Although we observed significant reductions in total cardiac output in aged animals (Figure 2B), the mechanical performance of each individual contraction was remarkably preserved. We observed no significant changes in stroke volume (Figure 2B, red), chamber diameter (Figure 2—figure supplement 1C), or fractional shortening (Figure 2—figure supplement 1D), suggesting that the observed decline in cardiac performance might reside in the temporal rather than spatial domain. Indeed, the most striking change associated with aging was an increased latency in transitioning from systole to diastole, which primarily reflected a decrease in the velocity of relaxation (positive values in Figure 2C) and an increase in the time period from peak contraction velocity to peak relaxation velocity (Figure 2D, blue, R2 = 0.73). In contrast, the kinetics of contraction was much less affected (Figure 2C,D). Similar diastolic decline with preserved contractile performance accounts for an increasing fraction of heart failure cases in humans (Borlaug, 2014; Sharma and Kass, 2014), suggesting a conserved susceptibility to aging-related declines in the structural or biochemical processes facilitating relaxation. A pair of K2P subunits, sandman and galene, are required for terminating systole in aged animals The rhythmic contraction and relaxation of the heart requires a precisely tuned series of ionic conductances that entrain the influx and efflux of calcium across the sarcolemnal and sarcoplasmic reticular membranes (Bers, 2008; Monfredi et al., 2013). In cardiomyocytes, outward potassium currents mediating repolarization are essential for terminating systole and suppressing dysrhythmic afterdepolarizations (Nerbonne and Kass, 2005). One physiological hallmark of failing hearts is the progressive loss of these repolarizing currents (Beuckelmann et al., 1993). We therefore investigated the repolarizing mechanisms maintaining diastolic function and normal rhythm in Drosophila. Previous work has implicated a number of potassium channels important in the repolarization to, or maintenance of, the cardiac resting potential in Drosophila, including KCNQ (Ocorr et al., 2007) and the K2P channel ORK1 (Lalevée et al., 2006). While several voltage-gated potassium channels have well established roles in cardiac repolarization, a number of K2P channel family members are expressed in the human heart but their physiological relevance remains an active area of investigation (Schmitt et al., 2014). TASK-1 (K2P3.1) has been implicated in chronic atrial fibrillation, where TASK-1 protein levels are increased and action potential duration is shortened in a TASK-1 dependent manner relative to controls (Schmidt et al., 2015). In the larval heart of Drosophila, the K2P family member ORK1 appears to fine tune the rate of slow diastolic depolarization (Lalevée et al., 2006). Transcriptional profiling has previously revealed a putative two-pore potassium channel subunit, CG8713, recently named sandman (Pimentel et al., 2016), with enriched mRNA expression in the Drosophila heart relative to other tissues (Robinson et al., 2013). Reasoning that this gene may play a role in cardiac repolarization, we confirmed its expression in the fly heart using RT-PCR and generated a small deletion of CG8713 and the adjacent gene CG8712 (Figure 3—figure supplement 1A,C). At 50 days of age, the hearts of sandman mutants displayed a marked inability to transition from heart contraction (systole) to heart relaxation (diastole), with some animals rarely or never displaying heart relaxation (Figure 3A, Video 2). Figure 3 with 2 supplements see all Download asset Open asset Diastolic failure in sandman mutants. (A) Representative YT kymographs of 50-day-old animals. Scale bars: (black vertical) 75 µm, (blue horizontal) 1 s. (B) Median heartbeat per genotype at 50 days of age. ⊕, clean excision of the mutagenic piggyBac insertion e00867. (C) Two-dimensional probability map of fractional shortening and systolic interval at 50 days of age. See also Video 2. https://doi.org/10.7554/eLife.20851.008 Figure 3—source data 1 Median heartbeats for all individual animals in panel B. Median heartbeats were calculated for individual animals (Table) and for all consolidated heartbeats for a respective genotype (Panel B and last column of each Table). This source data provides a representation of the observed heartbeat waveform variability between animals. https://doi.org/10.7554/eLife.20851.009 Download elife-20851-fig3-data1-v1.xlsx Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 50-day wild-type and sandman heartbeat visualization. One-third speed videos of 50-day adult wild-type (upper video) and sandman (lower two videos) females, with heart wall position calls (yellow) and the attending transformation into heart chamber diameter as a function of time in a 1 s streaming window. The initiation and end of each contraction are specified by a red and blue triangle, respectively. https://doi.org/10.7554/eLife.20851.012 This phenotype appears to be heart autonomous and specifically due to the loss of sandman, not CG8712. Expression of the sandman cDNA in cardiomyocytes using R94C02::Gal4 rescued the functional defects observed, including the median heartbeat waveform (Figure 3B) and the probability map of fractional shortening versus systolic interval (Figure 3C). We next screened other putative K2P channel subunits and uncovered a similar heart-autonomous role the heart expressed K2P subunit CG9194 (henceforth galene) (Figure 3—figure supplement 1B,C), for which knockdown also led to a marked decline in cardiac function. Heterozygous animals expressing dsRNA selectively targeting in the heart using displayed a in fractional shortening versus systolic interval (Figure 3—figure supplement a significant reduction in cardiac output per second and per stroke (Figure 3—figure supplement 2B), and a prolongation of the systolic interval relative to controls (Figure 3—figure supplement As in sandman this primarily a in transitioning from systole to the median heartbeat waveform a clear reduction of diastolic function relative to controls (Figure 3—figure supplement The sandman phenotype an progression in cardiac repolarization can manifest as life but their pathophysiology is due to compensatory repolarization and electrical remodeling (Nattel et al., 2007). atrial fibrillation exhibits an that remodeling and normal aging. The action potential is which further shortening of the action potential the aging hearts action potential which but also the heart to a ventricular rhythm that can to cardiac Such is evident in a Drosophila model of where KCNQ potassium channel mutants develop progressive (Ocorr et al., 2007). We the progression of diastolic failure in sandman mutants. sandman mutants displayed defects in cardiac function early in output per second and per stroke were from wild-type at 10 days of age (Figure with in the systolic interval (Figure However, by 30 days of age heart function was cardiac output per second and per stroke (Figure while the systolic and diastolic increased (Figure 50 days of age, median total cardiac output per second (Figure This progression can also be observed in two-dimensional probability of chamber diameter and fractional shortening (Figure supplement and can be rescued by expressing sandman cDNA selectively in cardiomyocytes of sandman mutants (Figure but not expressed selectively in wall the heart Figure supplements 2 and 3 for detailed for all measurements by age and Figure with 3 supplements see all Download asset Open asset heart failure in sandman mutants. Estimated cardiac output per second (A) and per stroke (B) were well by a and the systolic (C) and diastolic (D) were well by for wild-type sandman cardiomyocyte of sandman using R94C02::Gal4 and the clean excision at specified n = to animals per genotype and age. The shaded areas represent the mean +/− s.d., with the regressions plotted as lines. and a heteromeric potassium channel in Although currents that influence the resting potential of K2P channels are by diverse and may therefore the of tissues to their et al., 2010). sandman was recently implicated as a potassium that as of a homeostatic (Pimentel et al., We characterized the behavior of sandman and by expressing in with their similar in vivo, sandman and most likely two subunits that a heteromeric ion channel in subunit expressed was to to an in in outward currents at (Figure This is highly for potassium in contrast to the open ORK1 et al., outward that is due to by (Figure a conserved feature of many K2P channel et al., this functional appears the two most K2P subunits, and not functional with or (Figure supplement 1). of potassium channel subunits is a for increasing of potassium channels as well as K2P channels and that several K2P channel are as in et al., et al., heteromeric as observed between and (Figure may be of to the physiology of this Figure with 1 supplement see all Download asset Open asset sandman and a potassium (A) Representative currents in physiological and from = = and of = during (B) currents from in The dotted the for a ion channel with no in The the observed reversal potential to a at The solution is 3 10 The solution and or are as the solution which 2 for the n = All data represent the mean +/− All are relative to Figure data 1 data for panel B. Download The sandman phenotype likely a loss of repolarization rather than remodeling To our Drosophila represents the model where the pumping action of the heart is acutely dispensable for adult We therefore characterized the phenotype of sandman mutants to determine whether it may reflect structural remodeling or a heart that is To these we acutely the calcium into the of intact days sandman animals and the heart's contractile and robustly the observed in sandman establishing that cardiomyocytes were in a dependent manner rather than into remodeling (Figure of the potassium also the prolonged observed in sandman mutants (Figure that the phenotype was dependent on the intracellular of potassium and likely reflected a failure to