|
Fig. 1.
CDK9i treatment resolves neutrophil infiltration following cardiac injury by promoting reverse migration. (A) Experimental timeline indicating cardiac injury, CDK9i treatment and imaging time points. (B) Epifluorescence images of Tg(myl7:GFP;mpx:mCherry) larvae displaying neutrophil presence on the injured ventricle at 4 hpi (prior to treatment), and at 6 hpi and 24 hpi with 0.3% DMSO vehicle (top), 50 μM AT7519 (middle) or 3 μM FVP (bottom). Arrowhead indicates ventricular apex injury site marked by a loss of myocardial GFP and neutrophil accumulation. (C) Number of ventricular neutrophils at 4 hpi, 6 hpi and 24 hpi with 50 μM AT7519 (top) or 3 μM FVP (bottom) treatment. Error bars represent s.e.m., n=19 larvae, experimental n=3. ****P<0.0001 (two-way ANOVA and Bonferroni post-hoc test for comparisons between cardiac-injured DMSO vehicle or CDK9i treatment groups). (D) LSFM images of neutrophil (mpx:mCherry) migration temporally colour coded between 4 hpi and 6 hpi with 0.1% DMSO vehicle (top) or 50 μM AT7519 (bottom). Neutrophil positions appear as a different colour depending on the point in time (as indicated in the key). Dashed line indicates outline of ventricle. Coloured arrowheads indicate starting position of neutrophil (DMSO vehicle, blue arrowhead with white outline) or ending position of neutrophils (AT7519, blue and green arrowheads with white outline) relative to image panels in E and F, respectively. White arrowhead indicates ventricular apex injury site. (E) LSFM time-lapse-derived images of ventricular neutrophil migration with DMSO vehicle (0.1%); hpi indicated above each image. Blue arrowhead tracks an individual neutrophil migrating across the ventricular apex. (F) LSFM time-lapse-derived images of neutrophil migration from ventricle to pericardium with AT7519 (50 μM) treatment; hpi indicated above each image. Blue and green arrowheads track individual neutrophils reverse migrating anteriorly and posteriorly to the pericardium, respectively. LSFM fluorescence images were acquired in 3D and maximum intensity projections were used for temporal colour code analysis (D) or are individually displayed (E,F). Scale bars: 50 μm.