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. 2017 May 19;13(5):e1006786.
doi: 10.1371/journal.pgen.1006786. eCollection 2017 May.

Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations

Karen Ocorr  1 , Alexander Zambon  1 , Yoav Nudell  1 , Santiago Pineda  1 , Soda Diop  1 , Min Tang  1 , Takeshi Akasaka  1 , Erika Taylor  1
Affiliations

Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations

Karen Ocorr et al. PLoS Genet. .

Abstract

Understanding the cellular-molecular substrates of heart disease is key to the development of cardiac specific therapies and to the prevention of off-target effects by non-cardiac targeted drugs. One of the primary targets for therapeutic intervention has been the human ether a go-go (hERG) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, we show here that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. We conclude that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
(A) qPCR of isolated hearts and heads from wildtype (w1118) Drosophila. Seven potassium channels, KCNQ, seizure (sei), the BK channel slowpoke (slo), shaker (sh), the inward rectifier (Ir), ether a go-go like (elk) and Shaker like (shal) exhibited significant expression in isolated hearts. Expression in the head (mostly nervous tissue) is shown for comparison. (B) Δ Ct values of different voltage-activated K+ channels calculated from raw Ct values in the wild-type Canton-S (WtCS) background. Note that ΔCt values are inversely correlated with relative expression such that a channel like elk is weakly expressed while the Ca2+ channel, Ca-alpha1D (CAD), as well as K+ channels KCNQ and sei have relatively high levels of expression. (C-E) Functional analysis of sei mutants shows alterations in heart function. (C, left) Mean Diastolic Intervals (DI) were significantly increased in hearts from 1 week old sei heterozygotes and homozygote mutants compared to their genetic background control WtCS. (C, right) The increase in mean DI observed for seits1 mutants was rescued by insertion of two genomic copies of wildtype sei (seiwt2). Overexpression of an extra copy of wt sei in the wt background (+;seiwt1) did not affect DI. (D, left) Mean Systolic Intervals (SI) did not vary among the different genotypes. (D, right) Mean SI did not vary among the genotypes. (E, left) The incidence of arrhythmia (quantified as the heart period standard deviation normalized to the median heart period) was significantly increased in hearts from homozygous seits1 homozygous mutants. (E, right) The increase in arrhythmia was partially rescued by insertion of two copies of the wt sei gene (seiwt2). (F-H) Contractility is impaired in sei mutants. (F, left) Heart diameters during diastole were significantly smaller in one heterozygote and in both homozygous sei mutants compared to their background control, WtCS. (F, right) Heart diameters during diastole were significantly smaller in hearts from sei rescue and overexpression flies compared to seits1 mutant. (G, left) The diameters of the hearts during systole did not vary significantly between the sei mutant lines and their background control, WtCS. (G, right) Heart diameters during systole were significantly smaller in hearts from sei rescue and overexpression flies compared to seits1 mutant. (H, left) Fractional shortening is reduced significantly in both of the homozygous sei mutant lines (H, right) but was not rescued in hearts from flies expressing genomic seiwt. For all figures significance was calculated using one-way ANOVA and Dunnett’s multiple comparison post-hoc test.; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Numbers of hearts examined are shown in bars in G.
Fig 2
Fig 2. Heart and adult-specific knockdown of sei affects heart rhythmicity and contractility.
Heart-specific KD of sei was achieved by crossing flies with a UASsei RNAi construct to the heart-specific driver tinCΔ4 Gal4. Adult specific KD was achieved using a temperature-sensitive driver tub-gal80ts in combination with tinCΔ4 Gal4 to knock down sei specifically in the adult heart (see Materials and methods). (A&B) Heart-specific (tin and adult specific KD of sei causes a significant increases in both systolic (A) and Diastolic Intervals (B) compared to controls (tinCΔ4 driver/+, tub gal80ts; tinCΔ4 driver/+, and UASsei RNAi /+) at 3 weeks of age. (C) Heart-specific and adult specific KD of sei causes a significant increases in arrhythmia compared to controls (tinCΔ4 driver/+, tub gal80ts; tinCΔ4 driver/+, and UASsei RNAi /+) at 3 weeks. (D) The fractional shortening, an indicator of heart contractility, was significantly reduced in 3 week sei KD hearts compared to controls. Significance was calculated using a two-way ANOVA and Tukey’s post-hoc test; *p<0.05, **P<0.01,****p<0.0001; number of flies examined is shown in bars in D.
Fig 3
Fig 3. Cardiac contractility under loaded conditions.
(A-AHL) Movement traces (top) and corresponding M-mode (bottom from a WtCS heart under unloaded conditions (normal AHL). Note that there are distinct movement peaks produced by the contraction and relaxation of the heart wall that are used along with a movable threshold bar (horizontal blue line in A & B) to accurately parse a single contraction into shortening (SP), isometric (ISO) and lengthening phases (LP). (A-+20% Ficoll) Hearts beating for 30 min under load (AHL + 20% Ficoll 400) show a prolonged shortening phase (SP) and an obvious isometric contraction phase (ISO). (B) Hearts from seits1 mutants do not exhibit a prolonged ISO phase under load. (C) Shortening velocities were calculated as (Diastolic Diameter-Systolic diameter)/ Shortening Phase, SP) see Cammarato et al, 2015. Under unloaded conditions Seits1 mutants showed significantly reduced shortening velocities compared to WtCS as well as to WtPE and KCNQ370 (a K+ channel mutant). (D) The length of time that hearts maintained isometric contractions was significantly increased under Load (plus 20% ficoll) for all genotypes except for sei mutants (ns = not significant; a-p<0.0001 with WtCS and WtPE; b-p<0.05 with KCNQ370). For C&D data are shown as the mean, 2.5–97.5% confidence interval, Maximum and Minimum values. Significance in (C) was determined using a one way ANOVA and in (D) using a two-way ANOVA, both with Tukey’s multiple comparisons post-hoc test, **p<0.01, ***p<0.001. (WtCS n = 16, seits1 n = 23, WtPE n = 24, KCNQ370 n = 16).
Fig 4
Fig 4. Spontaneous action potentials from Drosophila myocardial cells.
Action potentials were recorded using intracellular recording techniques from fly hearts in which contractions had been blocked with 10uM blebbistatin. (A,C,E) Action potentials from myocardial cells in hearts from young flies (1–2 week old). (B,D,F) Action potentials from myocardial cells in hearts from old flies (4–6 weeks). (G) Superimposed action potentials from two wt lines (WtPE-red; WtCS-black) and seits1 mutant (blue) hearts. (H-J) Action Potential Duration (APD) was measured for single-peak APs at the point where the membrane voltage had repolarized to 10% (H), 50% (I) and 90% (J) of its maximum depolarized potential. (one-way ANOVA;*p<0.05; ** p<0.01).
Fig 5
Fig 5. Simultaneous recording of heart wall movements and intracellular action potentials.
M-mode traces (depicting heart wall movement in the y axis, top) were synchronized with intracellular recordings (showing spontaneous voltage changes, bottom) from the hearts of (A) a 1 week old w1118 wildtype fly (B) a 1 week old seits1 mutant (C) a 1 week old KCNQ370 mutant and (D) a 4 week old KCNQ370 mutant. (E) Simultaneous Intracellular / Optical Recording Setup-An intracellular recording from a single myocardial cell in the heart (Input 1) and the voltage trace generated by the optical recording software (Input 2) used to synchronize the optical and electrical recordings. An M-mode from the first four seconds of the movie is shown (top).
Fig 6
Fig 6. hERG antagonists E-4031 and Dofetilide cause bradycardia.
(A) Histogram of binned diastolic intervals from 12 WtCS hearts before and after exposure to 1μM E-4031 expressed as a percentage of total DIs (totals are show in white for T0 and black for records at 15 min after drug / vehicle exposure). (B) Distribution of DIs in hearts from 14 sei/hERG mutants before and 15 min after exposure to 1μM E-4031. (C) Distribution of DIs in hearts from16 WtCS before and 15 min after exposure to AHL vehicle. (D) Distribution of DIs in hearts from 15 sei/hERG mutants before and 15 min after exposure to AHL vehicle. (E) Average DIs before and after exposure to either 1μM E-4031 or vehicle. (F) Average DIs before and after exposure to either 1μM Dofetilide or DMSO vehicle. Plotted values are mean ± SEM; significance was determined using a two way ANOVA and Sidak’s multiple comparisons test; *** p<0.001; ****p<0.0001.
Fig 7
Fig 7. Actin and myosin staining of isolated hearts shows myofibrillar reorganization in seizure mutants.
(A) Z stacks from in situ hearts (1 week old) stained with phalloidin to show actin filaments (green). Both WtCS and KCNQ370 mutant hearts show distinct circumferential myofibrils that are tightly packed. Sei mutant hearts show gaps between myofibrils that are disorganized (double headed arrows indicate comparable chambers of the heart). In addition, valve structures appear to be compromised (arrow heads). (B) Higher magnification of one chamber of the heart (abdominal segment 4) stained for actin (green) and myosin (red). Myofibrils in the heart from a 3 week old WtCS fly (top) are circumferentially organized and tightly packed; the same region from a 3 week old seits1 mutant heart (bottom) that shows severe myofibrillar disarray and obvious gaps.
Fig 8
Fig 8. Morphological remodeling in seizure mutants.
(A) A full projection Z stack of a heart from a 1 week old sei mutant stained with phalloidin for F-actin is shown. (B) Hearts were scored for presence of gaps (*in A) or (C) longitudinally oriented myofibrils (double headed arrow in A). Note also the presence of very thin myofibrils (arrow head in A, for wt structures see Fig 6B). Ages are in weeks; numbers of hearts examined are indicated in each bar in B. Significance was determined using multiple T-tests comparing each mutant with its genetic background control at each age; **p<0.01, ***p<0.001.
Fig 9
Fig 9. Cardiac-specific sei KD causes myofibrillar disarray and thinning.
(A) Phalloidin staining of F-actin in hearts from 3 week old flies reveals the circumferential myofibrillar structure in control (tinCΔ4Gal4/+). Scale bar is 50μm. (B) Hearts for 3 week old KCNQ KD flies (tinCΔ4Gal4>UASKCNQ RNAi) also show a normal circumferential myofibrillar pattern. (C) Cardiac-specific KD of sei results in myofibrillar disarray and gaps (arrows). * denotes the position of ostia.
Fig 10
Fig 10. Larval heart morphology in sei mutants.
Hearts from wandering 3rd instar larva (just prior to pupation) were fixed and stained for F-actin (green) and myosin (red). The posterior abdominal heart is shown for a WtCS (A&B) and seits1 mutant (C&D) heart at 10x (top) and 25x (bottom). No obvious differences were observed for mutant hearts compared to wt at this late larval stage.
Fig 11
Fig 11. Altered gene expression and GO Functions in Sei and KCNQ mutant hearts.
(A) Isolated hearts from control and channel mutant flies were subjected to gene expression analysis. Heat map shows hierarchical clustering of 1812 probesets that were significantly up (red) or down (blue) regulated in sei mutant hearts compared to WtCS (Fold>2.0, P<0.05, each line shows results for a single probeset and each column is a replicate) and the corresponding effect in hearts from KCNQ mutants (compared to WtPE). (B) Significantly UP (red) and DOWN (blue) regulated Go Functions (Z score>2) identified in KCNQ370 mutant hearts are shown with the corresponding percent of category genes that were significantly altered. Z scores are shown at the end of all bars. (C) Significantly Down regulated Go Functions identified in sei mutant hearts. (D) Significantly UP regulated Go Functions (Z score>2) in sei mutant hearts are shown.
Fig 12
Fig 12. sei mutations affect wnt gene expression and interact genetically with pygo in the heart.
(A) Schematic diagram of the Wnt signaling pathway highlighting the individual components showing significant down (green) or up (red) regulation in hearts from sei mutants compared to its WtCS background control. (B) Diastolic intervals in seits1/ pygo double heterozygotes are significantly longer than for seits1 and pygo single heterozygotes. (C) Systolic intervals in seits1/ pygo double heterozygotes are significantly longer than for seits1 and pygo single heterozygotes. (D) Fractional shortening, a measure of contractility, is significantly reduced in seits1/ pygo double heterozygotes compared to seits1 and pygo single heterozygotes. Significance was determined by one way ANOVA and Tukey’s post-hoc test; **p<0.01, ***p<0.001.
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