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Figure 4
Hemodynamic forces control regeneration and myocardial reprogramming.
( A–D) Confocal microscopy imaging performed on vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( B) blebbistatin or ( C) tricaine treatment as well as ( D) the gata2a-/- mutant allele inhibit CM proliferation at 48 hpt (7 dpf) when compared to CM proliferation of ( A) ethanol-treated control hearts. White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( E) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that blebbistatin or tricaine treatment, and gata2a-/- defects block CM proliferation in injured hearts (n = 23, control EtOH; 21 MTZ EtOH; 9 control Blebb; 9 MTZ Blebb; 10 control Tric; 10 MTZ Tric; 9 control gata2a-/-; 16 MTZ gata2a-/-). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( F) Quantitation of the percentage of vmhc:mCherry-NTR ventricle-ablated hearts that display recovered ventricular tissue and contractility (black bars) at 96 hpt (9 dpf) shows that inhibiting contractility by blebbistatin or tricaine treatment as well as decreasing blood viscosity using gata2a-/- mutants impair heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( G–J) Confocal microscopy imaging of vmhc:mCherry-NTR; amhc:CreERT2; β-actin2:RSG hearts at 60 hpt (7.5 dpf) reveals that ( H) blebbistatin-treated, ( I) tricaine-treated, or ( J) gata2a-/- ventricle-ablated hearts exhibit reduced ability of genetically labeled atrial CMs (c-aGFP+) to contribute to the regenerating injured ventricle when compared to ( G) ethanol sham-treated hearts. ( G’–J’) Green channel – genetically-labeled c-aGFP+ atrial CMs. ( K) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs confirms the reduced atrial CM contribution to the regenerating injured ventricle in gata2a-/- mutant hearts as well as wild-type hearts treated with blebbistatin or tricaine (n = 12 ethanol; 7 blebbistatin; 7 tricaine; 9 gata2a-/-). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ/DMSO treatment. Dashed lines outline the heart. Bars: 50 μm. ( E) Mean + s.e.m. ANOVA; ( K) Mean + s.d. ANOVA; ( F) Total numbers, Binomial test (versus EtOH); ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 4.