Hand factor ablation causes defective left ventricular chamber development and compromised adult cardiac function

Figures

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  Fig 1. The Hand1LV-Cre transgenic allele recombines within the embryonic LV. A) Schematic of the mouse Hand1LV-Cre transgene, in which a 744bp Hand1 LV-specific enhancer and ~2.7Kb Hand1 endogenous promoter regulate an eGFPCre expression cassette. B-M) X-gal staining to detect the R26RlacZ reporter allele demonstrates that, like the Hand1eGFPCre knock-in allele (B, D, F, H, J, L), Hand1LV-Cre activity initiates in the LV (white arrows) between E8.5 and E9.5 (C, E). Cre-lineage cells are detectable within the LV at E10.5 (G, I) and E15.5 (K, M), but not within the epicardium (H, I; black arrowheads) or the outflow tract (H, I; black arrows). la–left atrium, ra–right atrium, lv–left ventricle, rv–right ventricle.

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  Fig 2. Hand1LV-Cre-mediated DTA activation results in LV cardiomyocyte cell death and hypoplastic LV, which, through cardiomyocyte proliferation, recovers fully by E16.5. A, B) TUNEL on E9.5 sections of Hand1LV-Cre(+);R26RlacZ/+ control (A) and Hand1LV-Cre(+);R26RlacZ/DTA embryos. Sections are counterstained with propidium iodide (PI). C, D) Quantification of the number of TUNEL-positive cells per heart in control and Hand1LV-Cre(+);R26RlacZ/DTA embryos at E9.5 (C) and E10.5 (D). E-H) Whole mount lysotracker staining detecting cell death confirms that conditional activation of R26ReGFPDTA/+ via intercross to Hand1LV-Cre causes pronounced LV cardiomyocyte death as assayed at E10.5 (white arrow; n = 3). White arrows denote TUNEL or lysotracker-positive cells in the LV. White arrowheads denote TUNEL or lysotracker-positive cells in the pharyngeal arches. I-P) X-Gal staining to detect the R26RlacZ reporter allele demonstrates that activation of the R26ReGFPDTA allele nearly completely ablates the Hand1LV-Cre lineage (black arrow) by E9.5 (I, J; n = 5); however, LV hypoplasia is not apparent until E10.5 (K, L; n = 3) and persists at E12.5 (M, N; n = 4). Despite DTA-mediated ablation of the…

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  Fig 3. Hand1LV-Cre-mediated ablation of Hand1 and Hand2 results in a dysmorphic LV, and a single ventricle phenotype perinatally. A-T) R26RlacZ reporter staining reveals that ablation of both Hand1 and Hand2 results in an abnormally thickened ventricular wall (R-T, white asterisks) that reduces the LV chamber lumen at E12.5, E14.5, and E17.5 (n = 4, 4, and 8, respectively).

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  Fig 4. Hand1LV-Cre-mediated Hand1;Hand2 CKOs have impaired cardiac function as adults. Mice at P56 reveal significant decreases in fractional shortening (A) and ejection fraction (B) in Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx mutants (n = 8) relative to Hand1LV-Cre;Hand1fx/+;Hand2fx/+ littermates (n = 8). Data are represented as mean ± standard error of mean.

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  Fig 5. Hand1LV-Cre-mediated Hand1;Hand2 CKOs display abnormal myocardial gene expression and trabecular proliferation in the LV. A, B, E, F, I, J) Section in situ hybridization of E11.5 hearts showing that the transcription factors Tbx20 and Hey2 are both expressed throughout both the presumptive compact myocardium and IVS, but excluded from the trabecular myocardium in control hearts (A, B). This sharp delineation between compact and trabecular myocardium is lost in the LVs of Hand1LV-Cre;Hand1fx/fx;Hand2fx/+ hearts (E, F). In Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx hearts, most of the trabeculae in the LV ectopically express compact myocardium markers, whereas the RV trabeculae do not (I, J; n = 4). C, D, G, H, K, L) Section in situ hybridization of E11.5 hearts showing that Bmp10 and Nppb are both expressed throughout the trabeculae, and are largely excluded from the compact myocardium in control hearts (C, D). In Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx hearts, Bmp10 and Nppb expression is downregulated within the trabeculae (K, L; n = 4). M-T) Immunohistochemistry for Mki67 on E11.5 Control (M-P) and Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx hearts (Q-T). Dashed lines delimit the boundary of trabeculae…

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  Fig 6. Hand1LV-Cre-mediated Hand1;Hand2 CKOs display abnormal expansion of ventricular septum markers into the LV. A-I) Section in situ hybridization of E11.5 hearts showing expression of the chemokine Cxcl12 (A, D, G), the secreted Wnt inhibitor Dkk3 (B, E, H), and the transcription factor Irx2 (C, F, I) in control (A-C), Hand1 heterozygous; Hand2 CKO (D-F), and Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx hearts (G-I; n = 4). Black arrowhead (H) indicates the expansion of the Dkk3 expression domain. Black arrow (I) indicates the expansion of the Irx2 expression domain. la–left atrium, ra–right atrium, rv–right ventricle. Asterisks denote the left ventricle.

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  Fig 7. DTA ablation rescues the lethality and cardiac dysfunction seen in Hand1LV-Cre Hand1;Hand2 CKOs. A-K) Section in situ hybridization showing expression of Bmp10 (A, E, I), Irx2 (B, F, J), Dkk3 (C, G, K) in E14.5 embryonic hearts, and X-gal-stained bisected P56 Control (D), Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx (H), and Hand1LV-Cre;Hand1fx/fx;Hand2fx/fx;R26RDTA-rescued hearts (L). Note that the RV of the heart in panel L has been mechanically damaged. (M, N) Echocardiographic analyses of P56 mice revealed restoration of ejection fraction (M) and fractional shortening (N) in Hand1LV-Cre(+);Hand1fx/fx;Hand2fx/fx;R26RDTA mice (n = 15) compared to Hand1LV-Cre(+);Hand1fx/fx;Hand2fx/fx CKOs. Hand1LV-Cre(-);R26RDTA mice, denoted as DTA(+), are included as controls. Data are represented as mean ± standard error of mean.