Thymosin beta 4 is dispensable for murine cardiac development and function

Abstract

Rationale: Thymosin beta 4 (Tβ4) is a 43-amino acid factor encoded by an X-linked gene. Recent studies have suggested that Tβ4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of tβ4 has been reported to result in embryonic lethality at E14.5-16.5, with severe cardiac and angiogenic defects. However, this shRNA tβ4-knockdown model did not completely abrogate Tβ4 expression. To completely ablate Tβ4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical.

Objective: We examined the role of Tβ4 in developing and adult heart through global and cardiac specific tβ4-knockout mouse models.

Methods and results: Global tβ4-knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global tβ4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific tβ4-deficient mice, generated by crossing tβ4-floxed mice to Nkx2.5-Cre and αMHC-Cre, were also found to have no phenotype.

Conclusions: We conclude that Tβ4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.

Figure 1. Generation of tβ4-knockout mice

(A) Targeting strategy, restriction map of genomic region of tβ4, in the middle is the targeted construct and at the bottom is the mutated loci after recombination. P1, P2 and P4 represent primer sites for genotyping. The grey rectangle denotes the targeted exon which is the 2nd exon encoding the majority of the coding region. Black triangles indicate LoxP sites, and black boxes indicate frt-NEO-frt cassettes. DTA, Diphtheria Toxic A chain gene; Neo, Neomycin resistance gene (B) Detection of wild-type and mutated alleles by Southern blot analysis. DNA from electroporated ES cells was digested with EcoR V and analyzed by Southern blot analysis with the probe diagrammatically represented in A. The 9.1 kb and 5.5 kb bands represent wild-type and mutated alleles, respectively. (C) PCR analysis of DNA isolated from Female (tβ4^+/+, tβ4^−/− ) and Male (tβ4^+/y and tβ4^−/y) mouse tails. (D) Real-time PCR confirmation of global gene deletion from Female (tβ4^+/+, tβ4^−/−) and Male (tβ4^+/y and tβ4^−/y) mouse hearts. (E) Western analyses of adult Male (tβ4^+/y and tβ4^−/y ) mouse hearts (H), Thymus (Th), Lung (Lu), Spleen (Sp), Brain (B). HA tagged Tβ4 isolated from COS cells used as control. Observed at approximately 4kDa. (D) Immunostaining from Heart and liver from E11.5 tβ4^Wt and tβ4^ko mouse hearts and liver. Tβ4 (Green), DAPI (Blue).

Figure 1. Generation of tβ4-knockout mice

(A) Targeting strategy, restriction map of genomic region of tβ4, in the middle is the targeted construct and at the bottom is the mutated loci after recombination. P1, P2 and P4 represent primer sites for genotyping. The grey rectangle denotes the targeted exon which is the 2nd exon encoding the majority of the coding region. Black triangles indicate LoxP sites, and black boxes indicate frt-NEO-frt cassettes. DTA, Diphtheria Toxic A chain gene; Neo, Neomycin resistance gene (B) Detection of wild-type and mutated alleles by Southern blot analysis. DNA from electroporated ES cells was digested with EcoR V and analyzed by Southern blot analysis with the probe diagrammatically represented in A. The 9.1 kb and 5.5 kb bands represent wild-type and mutated alleles, respectively. (C) PCR analysis of DNA isolated from Female (tβ4^+/+, tβ4^−/− ) and Male (tβ4^+/y and tβ4^−/y) mouse tails. (D) Real-time PCR confirmation of global gene deletion from Female (tβ4^+/+, tβ4^−/−) and Male (tβ4^+/y and tβ4^−/y) mouse hearts. (E) Western analyses of adult Male (tβ4^+/y and tβ4^−/y ) mouse hearts (H), Thymus (Th), Lung (Lu), Spleen (Sp), Brain (B). HA tagged Tβ4 isolated from COS cells used as control. Observed at approximately 4kDa. (D) Immunostaining from Heart and liver from E11.5 tβ4^Wt and tβ4^ko mouse hearts and liver. Tβ4 (Green), DAPI (Blue).

Figure 2. Global tβ4-loss does not cause embryonic lethality

(A) E14.5 Gross images of embryos and hearts, top and bottom respectively. No pathology was observed between conditions. Scale bar 1mm (B) Frozen H&E sections from tβ4^+/y and tβ4^−/y E14.5 hearts, images at 1x. (C&D) Representative immunofluorescence staining of (C) Left ventricular free wall and (D) Aorta of tβ4^+/y and tβ4^−/y mice E14.5. 40x CD31 (Green) α–SMA (Red) and DAPI (blue). (E) Quantification of CD31 positive area normalized to nuclei numbers. n=3-5. N/C= No significant change.

Figure 3. Analyses of Epicardial markers in the adult and embryonic heart

(A) Immunostaining of E15.5 hearts from both Male (tβ4^Wt and tβ4^ko) mouse hearts. DAPI (Blue), Wt1 (Green). (B&C) Real-time PCR analyses of tbx18 and wt1 in the adult Female (tβ4^+/+, tβ4^−/−) ,and Male (tβ4^+/y and tβ4^−/y) mouse hearts. N/C= No significant change.

Figure 4. Global tβ4-loss does not cause adult pathology, fibrosis, alter myocyte size or morphology

(A) 12 week old hearts from tβ4^+/+, tβ4^−/− , tβ4^+/y and tβ4^−/y hearts. (B) 1 × and 40× Parrafin H&E sections from tβ4^+/+, tβ4^−/− , tβ4^+/y and tβ4^−/y hearts Scale bars 2mm and 200μm. (C&D) Masson’s trichrome (C), and Picrosirius red stained 40X Images. Scale bars 200μm (E) Wheat Germ Agglutinin staining from left ventricular frozen sections, original magnification 40X. Scale bar 50μm. (F&G) Heart Weight (mg)/Tibia length (mm) (F) and Heart Weight (mg)/Body Weight (g) (G) from 12 week old mice. (H) Myocyte size from WGA stained sections, 100 random cells per field, quantification from 10 random fields per heart, n=3-10 hearts per experiment. N/C= No significant change.

Figure 5. Global tβ4-loss does not result in loss of capillary bed density

(A) Representative 3D reconstruction of the capillary bed and CD31 staining of the murine left ventricular free wall at 12 weeks. (B) Real-time PCR analyses of angiogenic cascade from 12 week–old. (C) Quantification of capillary bed analyses/vessel volume (μm³) and CD31 positive areas per field. n-3-4 hearts per experiment. N/C= No significant change.

Figure 6. Cardiac-specific tβ4-loss does not cause adult pathology, fibrosis, alter myocyte size or morphology

(A&B) 12 week old hearts from Male (tβ4^f/y and tβ4^{f/y;nkx2.5cre}) hearts morphological analyses HW/BW, HW/TL (C-F) Real-time PCR analyses of fetal gene expression in 12 week old hearts from Male (tβ4^f/y and tβ4^{f/y;nkx2.5cre}) hearts. (G&H) Real-time PCR analyses of fibrotic genes from 12 week old Male (tβ4^f/y and tβ4^{f/y;nkx2.5cre}) hearts. (I) H&E stained 12 week old hearts from tβ4^f/y and tβ4^{f/y;nkx2.5cre} hearts, 1X. Scale bar, 2mm (J) Masson Trichrome stained sections from 12 week old hearts from tβ4^f/y and tβ4^{f/y;nkx2.5cre} hearts, 40×. Scale bar, 200μm. n-3-4 hearts per experiment. N/C= No significant change.