Figure 5

The mechanosensitive channel Trpv4 regulates endocardial Notch activation and myocardial regeneration through Klf2a.

( A–D) Confocal imaging of  vmhc:mCherry-NTR ; klf2a:H2B-GFP hearts shows that  klf2a:H2B-GFP expression is activated in ( Bwild-type ( wt) ventricle-ablated hearts at 24 hpt (6 dpf) compared to ( A) control hearts; however, this activation is reduced in ( Dtrpv4-/- ventricle-ablated hearts. ( F–H) Confocal imaging of  vmhc:mCherry-NTR ; Tp1:d2GFP hearts further reveals that  Tp1:d2GFP is decreased in ( Gklf2a-/- and ( Htrpv4-/- ventricle-ablated hearts at 24 hpt (6 dpf) when compared to ( F) wild-type ( wt) hearts. ( E, I) Quantitation of the relative average fluorescence intensity confirms reduced injury-induced ( Eklf2a:H2B-GFP activation in  trpv4-/- ventricle-ablated hearts, and ( ITp1:d2GFP activation in  trpv4-/- and  klf2a-/- ventricle-ablated hearts when compared to wild-type ventricle-ablated hearts ( klf2a:H2B-GFP n = 9 control  wt; 7 control  trpv4-/-; 9 MTZ  wt; 10 MTZ  trpv4-/-. Tp1:d2GFP n = 15 control  wt; 18 control  trpv4-/-; 20 control  klf2a-/-; 22 MTZ  wt; 21 MTZ  trpv4-/-; 18 MTZ  klf2a-/). ( J–L) Whole-mount in situ hybridizations show that  notch1b is decreased in ventricle-ablated ( Kklf2a-/- (n = 2/11) and ( Ltrpv4-/- hearts (n = 6/15) at 24 hpt (6 dpf) when compared to ( J) wild-type hearts (n = 16/18). ( M–O) Confocal microscopy performed on  vmhc:mCherry-NTR ventricle-ablated hearts reveals that ( Nklf2a-/- and ( Otrpv4-/-ventricle-ablated hearts display reduced CM proliferative response when compared to ( M) wild-type ( wt) ventricle-ablated hearts at 48 hpt (7 dpf). White – anti-phospho-histone H3 immunostaining; red – anti-MF-20 immunostaining. Arrows point to proliferating CMs. ( P) Quantitation of anti-phospho-histone H3+ CMs in these hearts confirms that  klf2a-/- and  trpv4-/- hearts fail to increase CM proliferation after ventricle-injury (n = 15 each condition). Red bars – ventricle; green bars – atrium; dark bars – control sham-ablated hearts; light bars – ventricle-ablated hearts. ( Q) 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) supports that  klf2a-/- and  trpv4-/- mutants exhibit impaired heart regeneration. The number of fish analyzed for each condition is indicated above each column. ( R–T) Confocal microscopy imaging of  vmhc:mCherry-NTR;  amhc:CreERT2;  β-actin2:RSGhearts at 72 hpt (8 dpf) shows that ( Sklf2a-/-and ( Ttrpv4-/- ventricle-ablated hearts exhibit reduced contribution of genetically labeled atrial CMs (c-aGFP+) to the regenerating injured ventricle when compared to ( R) wild-type ventricle-ablated hearts. Green channel – ( R’–T’) genetically labeled c-aGFP+ atrial CMs. ( U) Quantitation of the percentage of ventricular area covered with c-aGFP+ CMs confirms that  klf2a-/-or  trpv4-/- hearts display reduced capacity to undergo injury-induced atrial-to-ventricular trans-differentiation during injury and regeneration (n = 13  wt; 8  klf2a-/-; 10  trpv4-/-). 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, I, P) Mean + s.e.m. ANOVA; ( Q) Total numbers, Binomial test (versus wild-type); ( U) Mean + s.d. ANOVA; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 5.

Expression Data
Genes:
Fish:
Condition:
Anatomical Terms:
Stage: Day 6

Expression Detail
Antibody Labeling
Phenotype Data
Fish:
Condition:
Observed In:
Stage Range: Day 6 to Days 7-13

Phenotype Detail
Acknowledgments
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