1. Pygopus Maintains Heart Function in Aging Drosophila Independently of Canonical Wnt SignalingClinical Perspective
by Min Tang, Wuzhou Yuan, Xiongwei Fan, Ming Liu, Rolf Bodmer, Karen Ocorr, and Xiushan Wu
Circ Genom Precis Med
Volume 6(5):
October 15, 2013
Copyright © American Heart Association, Inc. All rights reserved.

2. pygo knockdown causes heart dysfunction.
pygo knockdown causes heart dysfunction. (A), Heart period, (B) systolic interval, and (C) diastolic interval were measured for hearts from 1-week-old wild-type control Drosophila (hand4.2/+), flies with cardiac-specific pygo knockdown (hand>UAS-pygo-RNAi), pygo rescue (hand>UAS-pygo-RNAi;UAS-pygo), β-cat/arm knockdown (hand>UAS-arm-RNAi), TCF knockdown (hand>UAS-TCF-RNAi), and overexpression of dominant-negative (DN) TCF (hand>TCF-DN). Note the significant systolic interval and diastolic interval interval prolongation with pygo knockdown and the partial rescue with pygo overexpression. (D), Cardiac arrhythmia index and (E) contractility, quantified as fractional shortening. In all measures the pygo knockdown phenotype is more severe than that in response to arm/β-cat or TCF knockdown. Significance was determined using a 1-way ANOVA and Tukey multiple comparisons post hoc test (for normally distributed data: systolic interval, fractional shortening, and diameters) or Kruskal–Wallis with Dunn multiple comparisons test (for non-Gaussian data: heart period, diastolic interval, and arrhythmia index). Differences relative to the Hand4.2/+ control are indicated by individual asterisks; significance between experimental groups is indicated by the capped lines; *P<0.5, **P<0.01, and ***P< Sample size was 20 to 30 flies per genotype.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

3. pygo knockdown (KD) compromises structural integrity.
pygo knockdown (KD) compromises structural integrity. (A), Five-second M-modes from movies of control (hand4.2/+, top), pygo KD (middle), and pygo rescue (bottom) hearts, illustrating qualitative differences in heart function parameters: heart period (HP, horizontal blue line), diastolic interval (DI, horizontal black line), diastolic diameter (vertical black line), systolic interval (SI, horizontal red line), and systolic diameter (vertical red line). pygo KD (middle) causes long DIs between contractions, longer fibrillatory SIs, and wider systolic diameters. (B–D), Phalloidin staining (green) for sarcomeric actin filaments, immunofluorescent staining of Nmr (red) for cardiac nuclei, and β-integrin (pink) that indicates the cell–cell contact between bilateral cardiomyocytes. Anterior is up in all images. (B), Cardiomyocytes from wild-type controls contain densely packed and circumferentially organized myofibrils (ie, normal myofibrillar orientation is perpendicular to the axis defined by the cell–cell contacts between adjacent pairs of cardiomyocytes). (C), pygo KD causes longitudinal reorientation or (D) overall disorganization with gaps between myofibrils. (E), The percentage of cardiomyocytes with normal (blue), disorganized (red), or reoriented (green) myofibrils in control, pygo KD, pygo rescue, arm KD, TCF KD, and TCF-DN hearts (n=20–30 hearts per genotype).
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

4. pygo loss of function exacerbates age-associated cardiac dysfunction.
pygo loss of function exacerbates age-associated cardiac dysfunction. Heart period (A), systolic interval (B), diastolic interval (C), and arrhythmia (D) are increased by heart-specific pygo knockdown compared with controls (hand4.2/+ and pygo-RNAi/+) and are further exacerbated with age. (E), pygo knockdown hearts exhibit a dramatically increased prevalence of prolonged systoles (>0.4 seconds, indicative of unsustained fibrillations) and (F) diastoles (>1.0 seconds, asystolic events), and these phenotypes are exacerbated with age. (G), Contractility (fractional shortening) is reduced in 1- and 3-week-old pygo knockdown hearts. (H), Heart diameters during diastole (dashed lines) are unchanged in pygo knockdown hearts, but systolic diameters are increased significantly in response to pygo knockdown at 1 and 3 weeks. For each data point (ie, at 1, 3, and 5 weeks), 20 to 30 individual hearts from flies of the specified genotype were analyzed; data are displayed as mean±SEM. Significance at each age was determined using a 1-way ANOVA and Tukey multiple comparisons post hoc test (for normally distributed data: systolic interval [SI], fractional shortening, and diameters) or Kruskal–Wallis with Dunn multiple comparisons test (for non-Gaussian data: heart period, diastolic interval [DI], and arrhythmia index). Significant differences with respect to both pygo-RNAi/+ and hand4.2/+ controls are indicated by individual asterisks; capped lines represent differences between specified groups (*P<0.05, **P<0.01, and ***P<0.001).
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

5. Effect of pygo knockdown on cardiac morphology with age.
Effect of pygo knockdown on cardiac morphology with age. (A) and (B), Representative confocal stacks of phalloidin stained hearts from 1-week-old flies; (C and D) 3-week-old flies, and (E and F) 5-week-old adult flies (segments A2-A3). (A), (C), and (E), Wild-type hearts exhibit regular circumferential myofibrillar organization, except at 5 weeks where some age-despendent disorganization is apparent. (B), (D), and (F), Cardiac-specific pygo knockdown hearts show myofibrillar disorganization and gaps that becomes more severe with age.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

6. Relative strength of RNAi-mediated interference by pygo, β-cat/arm, and TCF on canonical Wnt signaling in the eye size assay.
Relative strength of RNAi-mediated interference by pygo, β-cat/arm, and TCF on canonical Wnt signaling in the eye size assay. (A), Expression of activated armadillo (UAS-arm*) driven by the eye-specific GMR-Gal4 (GMR>arm*/+) dramatically reduces the eye size. (B), Concomitant expression of pygo-RNAi partially rescues the arm*-induced small eye phenotype toward normal eye size. (C), Partial reversion of the rescue by additionally expressing a wild-type pygo transgene (GMR>arm*/pygo-RNAi;UAS-pygo) demonstrates pygo’s participation in canonical Wnt signaling–mediated reduction in eye size. In contrast to the partial rescue by pygo-RNAi (v100724), (D) Knockdown of TCF or (E) arm rescues the arm*-induced small eye phenotype much more robustly, indicating their potency in interfering with canonical Wnt signaling.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

7. Knockdown of β-cat/arm in a pygo heterozygous background.
Knockdown of β-cat/arm in a pygo heterozygous background. (A–E) Knockdown of β-cat/arm in a heterozygous pygo genetic background (hand4.2-Gal4/arm-RNAi; pygoS123/+; showed no significant enhancement in cardiac dysfunction compared with that seen in response to cardiac-specific β-cat/arm knockdown alone. (F), There was a small increase in diastolic diameters in the pygo heterozygote background relative to β-cat/arm knockdown alone. Significance was determined using a 1-way ANOVA and Tukey multiple comparisons post hoc test. For non-Gaussian data (arrhythmia index), the Kruskal–Wallis with Dunn multiple comparisons test was used; significant differences among groups are indicated by capped lines; *P<0.5, **P<0.01, and ***P< Each bar represents 20 to 30 individual hearts that were analyzed for the indicated heart function parameters; data are displayed as mean±SEM.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

8. pygo interacts genetically with CaMKII to regulate heart function.
pygo interacts genetically with CaMKII to regulate heart function. Heart function parameters were measured in 1-week-old Drosophila controls (hand4.2/+) with cardiac-specific CaMKII knockdown (hand4.2/UAS-CaMKII-RNAi), with pygo knockdown and with pygo/CaMKII double knockdown (hand4.2/UAS-pygo-RNAi;UAS-CaMKII-RNAi). (A), Heart period was prolonged in cardiac-specific pygo knockdown because of (B) prolonged systolic and (C) diastolic intervals. CaMKII KD alone had no effect on HP but in conjunction with pygo knockdown caused an even greater increase in heart period and diastolic interval. (D), The incidence of arrhythmias was increased as expected in pygo knockdown but not in CaMKII knockdown hearts. The combined knockdown showed a trend toward further dysfunction in heart rhythm. (E), Fractional shortening decreased significantly in pygo diastolic intervals, but the double knockdown combination did not show a further decrease. (F), Interestingly, there was a significant increase in systolic diameter in pygo knockdown hearts that was exacerbated in the double knockdown. Hearts from double knockdown flies in diastole tended to be slightly dilated, but this was not different from the single CaMKII knockdown. For each genotype (bar) between 20 and 30 individual hearts were analyzed; data are displayed as mean±SEM. Significance was determined using a 1-way ANOVA and Tukey multiple comparisons post hoc test (for normally distributed data: systolic interval, fractional shortening, and diameters) or Kruskal–Wallis with Dunn multiple comparisons test (for non-Gaussian data: heart period, diastolic intervals, and arrhythmia index). Data that are significantly different from all 3 control lines (ie, Hand 4.2/+, CaMKII RNAi/+, and pygo-RNAi/+) are indicated by individual asterisks; significant differences between experimental groups are indicated by the capped lines; *P<0.5, **P<0.01, and ***P<0.001.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.

9. Increased levels of Pygo1 in cardiac hypertrophy.
Increased levels of Pygo1 in cardiac hypertrophy. (A), Immunohistochemistry using α-pygo antibody and Diaminobenzidine shows Pygo1 protein expression in transverse sections of ventricles from sham-treated mice. (B), Pygo1 protein expression is increased in ventricles from isoproterenol-infused mice. (C), Quantification of Western blot analysis of hearts from sham (n=4) and isoproterenol-infused (n=4) mice shows significant increases in both Pygo1 and atrial natriuretic factor protein levels. Protein levels are expressed relative to β-actin and are displayed as the mean±SEM. **P<0.005.
Min Tang et al. Circ Cardiovasc Genet. 2013;6:
Copyright © American Heart Association, Inc. All rights reserved.