Overexpression of Kif1A in the Developing Drosophila Heart Causes Valvar and Contractility Defects: Implications for Human Congenital Heart Disease

Figures

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  Figure 1. Patient with left-sided heart defects associated with a t(2,8;q37,p11) balanced translocation: (A) Molecular mapping of the 2q37 translocation breakpoint by fluorescence in situ hybridization. The transcription initiation site of the KIF1A gene is downstream of the breakpoint region. Whole mount, transverse sections of explanted aortic valve leaflets from (B) the patient and (C) an age- and sex-matched unaffected control. (D,E) Higher magnification images of sections from explanted aortic valve tissue from the patient (D,D’’) and an age and sex-matched unaffected control (E,E’’). D and E: Hematoxylin-Eosin stain showing interstitial cells; D’ and E’: Masson’s Trichrome stain showing loss of interstitial cells accompanied by increased fibrosis (blue) in the patient; and D’’ and E’’: Von Kossa stain showing calcification (brown) in the patient.

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  Figure 2. Kif1A overexpression causes reduced contractility and increased myofibrillar disarray: (A) Mesodermal (Twist;24B-Gal4 and 24B-Gal4, and mef2-Gal4) and cardiac-specific (GMH5-Gal4) drivers were used to drive (left) Kif1A and (right) unc104 overexpression in the hearts of 3-week-old flies. In all cases, overexpression of mouse Kif1A or fly unc104 significantly reduced fractional shortening, a measure of cardiac contractility, compared to controls. Significance was determined using a one-way analysis of variance and Tukey’s multiple comparisons post hoc test; number of flies in each group is shown in Supplementary Table S1. (B) Hearts from 3-week-old adult flies stained for F-actin: Control hearts (24B-Gal4/+) show compact myofibrillar structure and well-defined ostia (inflow tracts, arrowhead) and valve (asterisk). Kif1A overexpression resulted in hearts with primarily thin and disorganized myofibrils within the cardiac myocytes with many gaps due to myofibrillar disarray. For orientation purposes, the location of the inflow tracts is indicated by arrowheads. Scale bar is 50 microns; anterior is to the left.

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  Figure 3. Kif1A overexpression causes valve remodeling: (A) F-actin staining with phalloidin of a control heart (mef2-Gal4/+) showing circumferential arrangement of myofibrils in the heart chambers and a denser myofibrillar arrangement in the 3 valve structures (indicated by arrows). Scale bar is 50 microns; anterior is to the left in all pictures. (B) In Kif1A overexpressing (OE) hearts (mef2-Gal4 > UAS Kif1A), the 2 identifiable valves (indicated by arrows) show reduced myofibrillar structure. (C) F-actin staining in a control heart (24B-Gal4/+) shows dense myofibrillar structure in the valve (dashed line circle). (D) Staining in Kif1A OE hearts (24B-Gal4 > UASKif1A OE) shows disorganized and loosely arranged myofibrils in the valve. Note overall reduction in F-actin containing myofibrils in Kif1A OE hearts in both Figure 3B,D. (E) Quantitation of identifiable valves in controls (24B-Gal4/+ and mef2-Gal4/+) and in F-actin stained hearts from Kif1A OE flies (24B > UAS KifAa and mef2 > UAS Kif1A): The number of hearts is indicated in each bar; ****p < 0.0001, Mann–Whitney test.

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  Figure 4. Kif1A overexpression increases collagen IV deposition: (A) Anti-pericardin (collagen IV homolog) staining reveals the extracellular matrix structure surrounding the cardiac tube in control flies expressing the muscle cell driver 24B-Gal4. (B) Muscle-specific Kif1A overexpression caused a significant increase in the extent of cardiac-associated collagen IV network. Scale bar is 50 microns; anterior is to the left. (C) Quantification of the ECM networks from Z-stacks shows significant increases in response to muscle cell-specific Kif1A OE. Significance was determined by two-tail, unpaired T-tests.

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  Figure 5. Actin cytoskeleton is affected in Kif1A-transfected C2C12 myoblasts: (A–E) Phalloidin staining reveals F-actin organization in transfected C2C12 cells. α−GFP or α−myc staining identifies cells transfected with (A’) AcGFP (controls), (B’) full-length myc-tagged Kif1A, (C’) myc-tagged Kif1A lacking the PH domain, (D’) myc-tagged Kif1A lacking the MD domain, and (E’) myc-tagged Kif1A lacking the FHA domain. The F-actin organization is displaced more towards the periphery of the cell (open arrowheads) where there are aggregates of Kif1A (solid arrowheads), as shown in Figure 5B,C. This effect is observed in the construct lacking the lacking the PH domain but not in those lacking the motor domain or the forkhead-associated domain, implying that normal motor domain function is required for overexpressed Kif1A protein to exert its effect. (F) Schematic illustrating the structure of full-length and truncated Kif1A proteins. MD: kinesin motor domain, FHA: forkhead associated domain, and PH: Pleckstrin homology domain. All the constructs listed in Figure 5F were tagged with myc for detection; (G) Kif1a transfection increases cell size. Two-dimensional cell surface area was measured in…

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  Figure 6. Kif1A overexpression impairs viability and cell polarity: Kif1A overexpression impairs viability of ommatidia and pigment cells. (A) One-week-old Kif1A x yw and (B) GMR-Gal4xyw controls. (C) Driven Kif1A OE in age-matched flies exhibited rougher and more decolorized external eyes compared to genetic controls. Kif1A overexpression impairs bristle arrangement. (D) One-week-old Kif1A x yw and (E) pnr x yw controls. (F) Overexpression of Kif1A in dorsal mesoderm using the Pnr-Gal4 driver resulted in disorientated bristle arrangement compared to genetic controls.

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  Figure 7. Kif1A overexpression affects muscle maturation: Twenty-four hours after transfection (with Kif1A-myc or AcGFP), C2C12 myoblast cells were switched to differentiation medium for 36 h and then fixed and stained. (A–A’’’) Kif1A transfected cells. (B–B’’) GFP-transfected cells. (A,B) Phalloidin staining reveals F-actin organization in all cells. (A’) Anti-myc staining to detect Kif1A expressing cells and (B’) anti-GFP staining to detect GFP-transfected control cells. (A”,B”) DAPI nuclear staining. (A’’’,B’’’) Merged images: note that differentiated C2C12 cells with Kif1A overexpression (A,C–C’”) formed a syncytium but contained scant cytoplasm.