Galactokinase is a novel modifier of calcineurin-induced cardiomyopathy in Drosophila

Abstract

Activated/uninhibited calcineurin is both necessary and sufficient to induce cardiac hypertrophy, a condition that often leads to dilated cardiomyopathy, heart failure, and sudden cardiac death. We expressed constitutively active calcineurin in the adult heart of Drosophila melanogaster and identified enlarged cardiac chamber dimensions and reduced cardiac contractility. In addition, expressing constitutively active calcineurin in the fly heart using the Gal4/UAS system induced an increase in heart wall thickness. We performed a targeted genetic screen for modifiers of calcineurin-induced cardiac enlargement based on previous calcineurin studies in the fly and identified galactokinase as a novel modifier of calcineurin-induced cardiomyopathy. Genomic deficiencies spanning the galactokinase locus, transposable elements that disrupt galactokinase, and cardiac-specific RNAi knockdown of galactokinase suppressed constitutively active calcineurin-induced cardiomyopathy. In addition, in flies expressing constitutively active calcineurin using the Gal4/UAS system, a transposable element in galactokinase suppressed the increase in heart wall thickness. Finally, genetic disruption of galactokinase suppressed calcineurin-induced wing vein abnormalities. Collectively, we generated a model for discovering novel modifiers of calcineurin-induced cardiac enlargement in the fly and identified galactokinase as a previously unknown regulator of calcineurin-induced cardiomyopathy in adult Drosophila.

Keywords: Drosophila melanogaster; calcineurin; cardiomyopathy; galactokinase.

Figure 1

tinC-CanA^act flies display a cardiac enlargement phenotype. (A) Domain structure of calcineurin, tinC-YFP-tagged CanA^act (tinC-YCanA^act), and tinC-Flag-tagged CanA^act (tinC-FCanA^act). CanA^act, constitutively active calcineurin; B, CanB binding domain; M, calmodulin binding domain; I, autoinhibitory domain. tinC is composed of a 304-bp genomic DNA element driving transgene expression in all cells constituting the heart tube as previously described (Yin et al. 1997; Lo and Frasch 2001; Wolf et al. 2006). (B) Representative OCT m-mode images and summary data for end-diastolic dimension and fractional shortening of w¹¹¹⁸ control (N = 16) and heterozygous tinC-YCanA^act from cross with w¹¹¹⁸ (N = 19) flies. CanA^act flies show enlarged end-diastolic dimensions and reduced fractional shortening. (C) Representative transverse paraffin sections of homozygous tinC-GFP control (N = 11) and tinC-YCanA^act (N = 9) fly hearts (blue arrows indicate the fly heart). Summary data for lumen area and perimeter are shown in micrometers. The tinC-YCanA^act fly displayed a significantly enlarged cardiac lumen area and perimeter. (Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001 compared to either w¹¹¹⁸ or tinC-GFP control. Data represent mean ± SEM.)

Figure 2

Molecularly defined deficiencies Df(3L)ED4416 and Df(3L)BSC130 rescued calcineurin-mediated abnormalities in adult flies. (A) Genetic map of deficiency stocks tested (adapted from Gbrowse, http://flybase.org/cgi-bin/gbrowse/dmel). Dashed lines indicate the suppressor region. Genes within the suppressor region are shown in the magnified view below. (Green bars, rescuing deficiencies; red bars, nonrescuing deficiencies; black bar, deficiency that causes cardiomyopathy on its own.) (B and C) Summary data for (B) end-diastolic dimension and (C) fractional shortening of w¹¹¹⁸ control (first column) tinC-YCanA^act alone and in the context of molecularly defined genomic deficiencies. All deficiencies were tested in the heterozygous states. (N = 17–51 for each group.) Two deficiency lines rescued the tinC-YCanA^act phenotype, narrowing down the original suppressor region to the region in between the dotted lines in A. Note that Df(3L)ED4421 covers the deficiency region but was dilated on its own without CanA^act expression (Figure S3) and was not considered in defining the deficiency region. (One-way ANOVA with Bonferroni correction; *P < 0.05, **P < 0.0001; data represent mean ± SEM.)

Figure 3

tinC-Gal4 > UAS-CanA^act displayed cardiac enlargement that was rescued by tinC-CanA^act-rescuing deficiencies and diminished contractility phenotype that was rescued by known calcineurin modifiers. (A and B) Summary data for (A) end-diastolic dimension and (B) fractional shortening of w¹¹¹⁸ controls (N = 9); tinC-Gal4 > UAS-YCanA^act (N = 32) alone; heterozygous tinC-Gal4 (N = 7); and tinC-Gal4 > UAS-YCanA^act in the presence of Mef2 (N = 11), sty (N = 12), CanB2 (N = 23), Df(3L)ED4416 (N = 18), and Df(3L)BSC130 (N = 13). All transgenes and mutations were heterozygous. Known modifiers of calcineurin signaling, Mef2, sty, and CanB2, rescued tinC-Gal4 > UAS-YCanA^act cardiac contractility (sty rescued fractional shortening but not end-diastolic dimension). In addition, the tinC-YCanA^act-rescuing deficiencies Df(3L)ED4416 and Df(3L)BSC130 also rescued tinC-Gal4 > UAS-YCanA^act cardiac enlargement [Df(3L)BSC130 rescued end-diastolic dimension but not fractional shortening]. (*P < 0.05, **P < 0.01, ***P < 0.0001; one-way ANOVA with Bonferroni correction; data represent mean ± SEM.)

Figure 4

Genetic disruption of Galk rescued tinC-CanA^act-induced reduction in cardiac contractility and tinC-Gal4 > UAS-CanA^act- induced hypertrophy. (A and B) Summary data for (A) end-diastolic dimension and (B) fractional shortening of flies expressing w¹¹¹⁸ control, tinC-YCanA^act, the transposable elements PBac{PB}Galk^c03848, Mi{ET1}Galk^MB10638, or precise excision of Mi{ET1}Galk^MB10638 (Mi{ET1}Galk^rev), in the context of tinC-YCanA^act. The two transposable elements in Galk rescued tinC-YCanA^act -mediated cardiac contractility (fractional shortening), while a precise excision reverted the rescue. [(A) *P < 0.05, **P < 0.01; (B) *P < 0.05, **P < 0.001, ***P < 0.0001 compared to tinC-YCanA^act or Mi{ET1}Galk^MB10638 as indicated with an overbar; N = 14–27 for each group; one-way ANOVA with Bonferroni correction for multiple comparisons, all data represent mean ± SEM.] (C and D) Summary data for (C) end-diastolic dimension and (D) fractional shortening of w¹¹¹⁸ control, CanA^act only, UAS-Galk no driver (heterozygous with w¹¹¹⁸), tinC-Gal4 driver only (heterozygous with w¹¹¹⁸), Galk RNAi only flies, and flies expressing tinC-YCanA^act in the context of cardiac Galk RNAi. RNAi to Galk significantly rescued tinC-YCanA^act-mediated cardiac enlargement. (*P < 0.01, **P < 0.0001 compared to tinC-YCanA^act; N = 7–20 for each group; one-way ANOVA with Bonferroni correction for multiple comparisons, all data represent mean ± SEM.) (E) qRT-PCR for Galk expression of w¹¹¹⁸ control, homozygous PBac{PB}Galk^c03848, or homozygous Mi{ET1}Galk^MB10638. Galk expression is downregulated by transposable element insertions. (*P < 0.001 compared to w¹¹¹⁸ control; N = 3 in each group; one-way ANOVA with Bonferroni correction for multiple comparisons, all data represent mean ± SEM.) (F) qRT-PCR of Galk expression in w¹¹¹⁸ control flies, flies heterozygous for tinC-YCanA^act alone, and flies in the context of heterozygous Mi{ET1}Galk^MB10638 or a precise excision of Mi{ET1}Galk^MB10638, Mi{ET1}Galk^rev. (*P < 0.05, **P < 0.001 compared to heterozygous tinC-YCanA^act/Mi{ET1}Galk^MB10638; N = 8 in each group; one-way ANOVA with Bonferroni correction for multiple comparisons, all data represent mean ± SEM.) (G) Galk expression for tinC-Gal4 heterozygous with w¹¹¹⁸ (driver only) and heterozygous UAS-Galk RNAi knockdown with a tinC-Gal4 driver (Galk RNAi). Galk expression is downregulated by transposable element insertions. (N = 3 for each group; Student’s t-test *P < 0.001, Galk RNAi vs. tinC-Gal4 driver only.) (H) Representative hematoxylin- and eosin-stained histological sections and quantification of wall thickness for heterozygous tinC-Gal4 (from cross with w¹¹¹⁸, N = 8), tinC-Gal4 > UAS-YCanA^act (N = 13), or tinC-Gal4 > UAS-YCanA^act in the context of Mi{ET1}Galk^MB10638 (N = 6). tinC-Gal4 > UAS-YCanA^act caused a cardiac hypertrophy phenotype that was rescued by Mi{ET1}Galk^MB10638. Blue arrowheads point to the heart. *P < 0.05, one-way ANOVA with Bonferroni correction, all data represent mean ± SEM. (All transgenes were heterozygous unless otherwise noted.)

Figure 5

The deficiency Df(3L)ED4416 and Minos insertion in Galk Mi{ET1}Galk^MB10638 rescued e16E-Gal4 > UAS-YCanA^act -induced wing vein loss. Wing vein phenotypes of heterozygous e16E-Gal4 driver only control or e16E-Gal4 > UAS-YCanA^act flies: normal, abnormality of the posterior crossvein (PCV), or abnormality of both PCV and longitudinal vein 5 (L5). Progeny were counted from the cross e16E-Gal4 × UAS-YCanA^act/CyO;Df(3L)ED4416/MKRS or e16E-Gal4 × UAS-YCanA^act/CyO;Mi{ET1}Galk^MB10638/MKRS. The graph represents percentage of all heterozygous e16E-Gal4 > UAS-YCanA^act flies counted with normal or abnormal wings in the context of heterozygous MKRS balancer (no deficiency), Df(3L)ED4416, or Mi{ET1}Galk^MB10638. Df(3L)ED4416 partially rescues the e16E-Gal4 > UAS-YCanA^act-induced wing vein loss phenotype [no deficiency, N = 46; Df(3L)ED4416, N = 54]. Mi{ET1}Galk^MB10638 also partially rescued the e16E-Gal4 > UAS-YCanA^act -induced wing vein loss phenotype (no Minos, N = 129; Mi{ET1}Galk^MB10638, N = 242). (Arrowheads point to the shortened abnormal PCV or L5, respectively. Significant rescue of the wing vein abnormality with Df(3L)ED4416 and Mi{ET1}Galk^MB10638 was determined by Fisher’s exact test, *P < 0.0001.)

Figure 6

The deficiency Df(3L)ED4416 did not rescue Act5C-Gal4 > UAS-YCanA^act-induced lethality, dpp-Gal4 > UAS-YCanA^act -induced unexpanded wing, or Mef2-Gal4 > UAS-YCanA^act-induced pupal lethality. (A) Progeny number from the cross Act5C-Gal4/CyO × UAS-YCanA^act/CyO;Df(3L)ED4416/TM2. Driving UAS-YCanA^act with an actin (Act5C-Gal4) driver resulted in a lethal phenotype, which was not rescued by Df(3L)ED4416. (B) Percentage of total abnormal-winged progeny from the cross dpp-Gal4/TM6B × UAS-YCanA^act/CyO;Df(3L)ED4416/TM2. Expressing CanA^act with a dpp driver resulted in a shriveled abnormal wing phenotype. This was not rescued by Df(3L)ED4416. (C) Total progeny from the cross Mef2-Gal4 × UAS-YCanA^act/CyO;Df(3L)ED4416/MKRS or Mef2-Gal4 × UAS-YCanA^act/CyO;Mi{ET1}Galk^MB10638/MKRS. Expressing CanA^act with a Mef2- driver resulted in a pupal lethal phenotype that was not rescued by Df(3L)ED4416 or Mi{ET1}Galk^MB10638. Data were analyzed using Fisher’s exact test.