Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart (original) (raw)
Early and late inactivation of Fog2 in fetal myocardium. To analyze the expression of Fog2 during heart development, we studied Fog2Lz/+ embryos, in which the LacZ reporter gene was driven by Fog2 regulatory elements. X-gal staining showed that Fog2 was robustly expressed in the heart and proepicardium from E9.5 to E11.5 (Figure 1A). In sections, we detected Fog2 expression in atrial and ventricular myocardium, epicardium, endocardium, and endocardial cushions (Figure 1B). As Fog2 was expressed in these different lineages, and previous work showed that transgenic Fog2 expression in cardiomyocytes only partially rescued the cardiac defects of Fog2–/– embryos (11), we sought to further define the tissue-restricted requirement of Fog2 for normal heart morphogenesis.
Temporal tissue-restricted inactivation of Fog2. (A) Whole-mount X-gal staining of E9.5–E11.5 Fog2Lz/+ embryos. (B) Section of an E10.5 Fog2Lz/+ embryo stained with X-gal. Fog2 was expressed in cardiomyocytes (1, red arrows), epicardial cells (black arrows), endocardial cells (green arrows), and AV cushion mesenchymal cells (2, asterisk). (C and D) Immunohistochemical staining for FOG2 (red) and NKX2-5 (green; cardiomyocyte marker). At E9.5, FOG2 was readily detected in Fog2fl/– control cardiomyocytes (yellow arrowheads). In littermate Fog2NK heart, FOG2 immunoreactivity was lost in myocardium (My, green arrowheads) but remained in endocardial cushion (Cu, white arrowheads). Staining of Fog2NKRosa26fsLz/+ for Cre-activated β-gal expression (red) confirmed recombination in cardiomyocytes at E9.5 (green; white arrows). (E and F) In Fog2MCRosafsLz/+ and Fog2fl/– hearts, at E9.5 FOG2 (red) was expressed in cardiomyocytes (green NKX2-5 staining; yellow arrowheads). At this stage, the Myh6-Cre transgene has not yet catalyzed efficient recombination, as indicated by lack of robust β-gal expression from the Cre-activated reporter (green arrows). (G and H) At E12.5, FOG2 immunoreactivity was readily detected in Fog2fl/– control cardiomyocytes (yellow arrowheads) but not Fog2MC mutant cardiomyocytes (green arrowheads). The Cre-activated reporter robustly expressed β-gal in cardiomyocytes at E12.5 (white arrows). Scale bar: 50 μm. All box regions indicate the magnified figures on right sides (C–H).
In order to study the temporal and spatial requirements for Fog2 in heart development, we took a conditional loss-of-function approach, using a floxed Fog2 allele (Fog2fl) (14). Recombination of Fog2fl by Cre recombinase deleted exon 8, yielding a nonfunctional allele that did not express FOG2 protein (14). We achieved early cardiomyocyte-restricted deletion of Fog2 using Cre recombinase expressed from NK2 transcription factor related, locus 5 regulatory elements (Nkx2-5Cre). (15). Nkx2-5Cre catalyzed efficient recombination of floxed targets in cardiomyocytes by E9.5 (6, 15). While FOG2 was readily detectable in cardiomyocytes of Fog2fl/–Nkx2-5+/+ control embryos (Figure 1C), FOG2 was not detectable in cardiomyocytes by E9.5 in Fog2fl/–Nkx2-5Cre/+ (Fog2NK) embryos (Figure 1D). FOG2 was not significantly recombined in endocardium or AV cushion mesenchymal cells of Fog2NK embryos, consistent with cardiomyocyte-restricted recombination of Fog2fl by Nkx2-5Cre (Figure 1D). Cardiomyocyte recombination by Nkx2-5Cre was confirmed using the Rosa26fsLz/+ reporter, which expresses β-gal only after Cre-mediated recombination. In Fog2NK embryos that also carried this reporter, β-gal was expressed in cardiomyocytes by E9.5 (Figure 1D). Cardiomyocyte Fog2 inactivation in Fog2NK hearts was also confirmed by immunohistochemistry at E12.5 (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI38723DS1).
In order to inactivate Fog2 at a later developmental stage, we used the Myh6-Cre transgene, in which Cre recombinase is driven by the cardiomyocyte-specific α-MHC promoter. Robust recombination in Myh6-Cre embryos occurred at E11.5 to E12.5 (6, 16). FOG2 immunostaining at E9.5 and E12.5 delineated the spatiotemporal pattern of Fog2fl recombination by Myh6-Cre (Figure 1, E–H). FOG2 expression was not significantly different between Fog2fl/–Myh6-Cre (Fog2MC) and littermate Cre-nontransgenic control (Fog2fl/–NTg) embryos at E9.5 (Figure 1, E and F). However, at E12.5, Fog2MC embryos displayed cardiomyocyte-restricted loss of FOG2 (Figure 1H). The later activity of Myh6-Cre was confirmed using the Rosa26fsLz/+ reporter in Fog2MC embryos. At E9.5, β-gal expression was not detectable in the Fog2MC heart (Figure 1F), while at E12.5, Fog2 was inactivated and β-gal was robustly expressed in most cardiomyocytes (Figure 1H).
Early cardiomyocyte-restricted ablation of Fog2 in Fog2NK embryos resulted in lethality by E13.5–E14.5. Fog2NK embryos developed subcutaneous edema, hemorrhage, and pericardial effusion, suggesting severe impairment of cardiac function (Figure 2, A and B). Fog2NK hearts had a constellation of defects that largely recapitulated the abnormalities seen in _Fog2_-null hearts (10, 11), including large atrial septal defect and VSD, AV endocardial cushion defect, thin compact myocardium, and overriding aorta (Figure 2, C–H). The coronary vascular plexus of Fog2NK mutants was dramatically decreased compared with that of littermates with any of the 3 control genotypes (Figure 2, I–K, and Supplemental Figure 2). To further corroborate the above findings, we visualized the coronary plexus of Fog2NK mutant and control embryos in which a LacZ reporter gene was driven by the endothelial-restricted Tie2 promoter (Tie2-Lz) transgene, which drives β-gal expression in endothelial cells (Figure 2, L and M). Control embryos demonstrated normal growth of the compact myocardium and multiple intramyocardial coronary vessels (Figure 2L). In contrast, Fog2NK hearts had very thin compact myocardium that contained few coronary vessels (Figure 2M).
Abnormal heart development and coronary vasculogenesis after early cardiomyocyte-restricted Fog2 inactivation. (A and B) Control (Fog2fl/+Nkx2-5Cre/+) and littermate Fog2NK mutant embryos at E13.5. (C–H) Representative H&E staining of Fog2NK (right panels) and littermate control (left panels; Fog2fl/+Nkx2-5Cre/+) hearts at different levels. RA, right atrium; LA, left atrium; RV, right ventricle; A, aorta. The asterisk indicates AV cushion and the arrow indicates VSD. (I–K) PECAM whole-mount staining of Fog2NK and control (Fog2fl/+Nkx2-5Cre/+) hearts at E13.5. The coronary endothelial plexus coverage (ratio of region within black dotted line to total projected ventricular area) was significantly decreased in mutants. (*P < 0.05; n = 4–5 per group as indicated.) (L) Section of X-gal stained Tie2-Lz hearts showed decreased coronary vessels in the compact myocardial layer of Fog2NK mutant compared with Fog2fl/+Nkx2-5Cre/+ littermate control. Scale bar: 500 μm. Ventricles in the boxed regions are magnified (right panels).
To study the function of myocardial Fog2 at later time points, we examined cardiac anatomy and coronary vascular development of Fog2MC embryos at E14.5. H&E staining of heart sections did not show structural heart defects in Fog2MC embryos (Supplemental Figure 3, A and B). The coronary vascular plexus formed normally, as demonstrated by whole-mount staining for the endothelial marker PECAM (Supplemental Figure 3, C and D). Sections of whole-mount stained hearts showed that the compact myocardium thickened normally and contained a normal number of intramyocardial coronary vessels (Supplemental Figure 3, E and F). Consistent with normal cardiac morphogenesis and coronary vascular development, Fog2MC mice survived to weaning at the expected Mendelian frequency (Table 1).
Tissue-restricted Fog2 knockout
Fog2 deletion in epicardium, endocardium, and neural crest-derived cells. In addition to cardiomyocytes, several additional lineages are required for cardiac morphogenesis, including the epicardium, endocardium, and cardiac neural crest (17, 18). To determine whether Fog2 is required in these lineages, we used a panel of Cre alleles to inactivate Fog2 in each (Table 1).
To inactivate Fog2 in the epicardium, we used Cre knocked into the Wilms tumor 1 (Wt1) locus (Wt1GFPCre/+) (19). WT1 and Cre expression was restricted to the epicardium (Supplemental Figure 4, A and B). Epicardium-derived cells (EPDCs), labeled using Wt1GFPCre, were found within the myocardium (Supplemental Figure 4C). These EPDCs have been implicated in coronary vessel development (20). However, epicardial deletion of Fog2 by Wt1GFPCre/+ did not impair coronary vascular development (Supplemental Figure 4D), suggesting that Fog2 is dispensable in epicardium and EPDCs for this process. However, Fog2fl/–Wt1GFPCre/+ fetuses developed severe AV endocardial cushion defects and thin myocardium at later developmental stages (Supplemental Figure 4E), and pups died in the perinatal period (Table 1 and data not shown). These data indicated that Fog2 expression in epicardium or its derivatives is required for normal growth and development of the AV valves and the compact myocardium.
To inactivate Fog2 in the endocardium, we used a Tie2Cre transgene to drive endothelial-restricted recombination. Tie2Cre inactivation of Fog2 (Fog2T2) did not detectably impact coronary plexus, endocardial cushions, or compact myocardium development (Supplemental Figure 5, A and B), and the survival rate to weaning was normal (Table 1). Adult Fog2T2 mice had normal tricuspid and mitral valves (Supplemental Figure 5C). Functional redundancy with Fog1 in endothelial-derived cells may contribute to the lack of phenotype in Fog2T2 mice (21).
To investigate the functional requirement of Fog2 in cardiac neural crest, we used the Wnt1-Cre transgene (22), in which wingless-related MMTV integration site 1 (Wnt1) regulatory elements drive Cre expression in cardiac neural crest. Fog2fl/–Wnt1-Cre mice survived normally to weaning and had no detectable defect in either heart morphogenesis or coronary development (Table 1 and data not shown).
Epicardial epithelial-to-mesenchymal transition is normal in Fog2–/– heart. Through an epithelial-to-mesenchymal transition (EMT), epicardial cells generate mesenchymal cells, which migrate into the subjacent myocardium (Supplemental Figure 4C) (19, 23, 24). Because epicardium and epicardial EMT are necessary for coronary vasculogenesis (20, 23), it was hypothesized that the coronary vascular defect of _Fog2_-null mutants was due to aborted epicardial EMT (11). To test this hypothesis, we first assessed the competence of _Fog2_-null epicardium to undergo EMT. When E12.5 Fog2+/+ embryo heart apexes were cultured on a collagen gel, epicardium grew off the explant as an epithelial sheet. At the edge of this cobblestone-like epithelial sheet, a subset of cells delaminated from the sheet, adopted a spindle-like shape, migrated into the collagen gel, and expressed the mesenchymal marker SMA (Figure 3, A–C). The behavior of Fog2–/– explants was not significantly distinguishable from Fog2+/+ explants in this assay (Figure 3D), indicating that Fog2–/– epicardial cells remain competent to undergo EMT.
Normal EMT in Fog2 mutant epicardium. (A) Representative figure of ex vivo heart explant on collagen gel. Cells transitioned to spindle-shaped mesenchymal cells (red arrows) at the edge of the epicardial outgrowth, and some delaminated from the epithelial sheet and migrated into collagen gel (red arrowheads). Inset shows an overview of the explant. (B) SMA staining (green) showed that most mesenchymal cells are at the edge of epicardial sheets. (C) A stack of confocal images of an explant cultured on a collagen gel, extending from the surface to the interior of the collagen gel, was acquired. Three-dimensional rendering showed migration of SMA+ cells into the gel. (D). Quantitation of the SMA cell number. EMT was not significantly different between Fog2–/– and Fog2+/+ explants. n = 4. (E) Experimental outline of tracing the fate of EPDCs in Fog2 mutants. Embryos were collected at E13.5. (F) Representative immunostaining for β-gal+ EPDCs in Fog2–/– and Fog2+/+ hearts. (G) Lack of significant difference between number of β-gal+ EPDCs in mutant compared with control (n = 3). Scale bar: 50 μm.
Next, we asked if epicardial EMT occurs normally in Fog2–/– embryos. Epicardium was intact in Fog2–/– embryos, as demonstrated by staining for epicardium-specific markers WT1 and RALDH2 (Supplemental Figure 6). Therefore, we used Cre-loxP lineage tracing to follow the fate of epicardial cells in Fog2+/+ compared with Fog2–/– hearts (Supplemental Figure 4C). Wt1GFPCre selectively activated Rosa26fsLz in the epicardium and its derivatives (Figure 3E), labeling EPDCs with β-gal. In Fog2+/+Wt1GFPCre/+Rosa26fsLz/+ hearts, β-gal+ EPDCs were observed within the myocardium (Figure 3F). In Fog2–/–Wt1GFPCre/+Rosa26fsLz/+ hearts, there was no significant difference in the number of β-gal+ EPDCs (Figure 3, F and G), indicating that epicardial EMT occurred normally in Fog2 knockout embryos. Taken together, our data show that the coronary vasculogenesis defect in Fog2 mutant embryos is not due to impaired epicardial EMT.
Fog2 is required for adult mouse survival and normal cardiac function. While Fog2MC mice survived normally to weaning, they died at 8–12 weeks of age (Figure 4A) with signs and symptoms of heart failure, indicating that FOG2 is required for normal adult heart function. Efficient inactivation of Fog2 was confirmed by quantitative RT-PCR (qRT-PCR) (Figure 4B). Echocardiography demonstrated that Fog2MC hearts were dilated and had severely depressed fractional shortening, a measure of systolic contractile function (Figure 4C and Table 2). Ventricular dilatation and depressed contraction were also demonstrated by cardiac imaging via PET (Supplemental Videos 1 and 2). On histopathological examination, Fog2MC hearts displayed cardiomegaly, wall thinning, and ventricular dilation (Figure 4, D and E). At an ultrastructural level, however, sarcomere and mitochondrial structure were preserved in Fog2MC cardiomyocytes (Supplemental Figure 7, A and B), suggesting that the striking Fog2MC phenotype is not attributable to gross sarcomere disorganization or mitochondrial abnormalities.
Loss of Fog2 leads to postnatal heart failure. (A) Kaplan-Meier survival curve of Fog2MC animals (n = 6 per group). (B) qRT-PCR showed efficient Fog2 knockout. (C) M-mode measurement of ventricular function in Fog2MC and littermate Cre-nontransgenic control (Fog2fl/–NTg). Fractional shortening (FS) was markedly reduced in Fog2MC compared with controls. (D and E) Gross view and sections of Fog2MC and littermate control Fog2fl/–NTg hearts, indicating dilated cardiomyopathy. The black bar indicates the compact myocardium. Scale bar: 2 mm. (F) qRT-PCR of heart failure-related genes in Fog2MC (red bars) and control (black bars). n = 8. (G) Representative TUNEL staining of Fog2MC heart section. Cardiomyocytes were identified by actinin staining. Arrows indicates apoptotic cardiomyocytes. The apoptotic cardiomyocyte in boxed region is magnified (lower-right panel). Scale bar: 10 μm. (H) Quantitation of TUNEL+ cardiomyocytes (n = 3 hearts per group). (I) Quantitation of activated caspase-3+ (aCasp3) cardiomyocytes (CM) (n = 3 hearts per group). *P < 0.05.
Echocardiographic parameters
Consistent with the heart failure phenotype, natriuretic peptide precursor type A (Anf, also known as Nppa) and natriuretic peptide precursor type B (Bnp, also known as Nppb) mRNAs, molecular markers of heart failure, were upregulated in Fog2MC hearts (Figure 4F). Although FOG2 was recently reported to regulate the sarcoplasmic reticulum Ca2+-ATPase (Serca2a, also known as ATP2a2) (25), Serca2 expression was not significantly changed in Fog2MC hearts (Figure 4F) or in neonatal Fog2fl/fl cardiomyocytes depleted of FOG2 by treatment with Cre adenovirus (data not shown). Gata4 expression was also not significantly changed in Fog2MC hearts (Figure 4F), indicating that heart failure in this model was not due to epistatic changes in Gata4 expression.
Increased cardiomyocyte loss from apoptosis has frequently been observed in heart failure, and cardiomyocyte apoptosis can, by itself, cause cardiac failure (26, 27). To better understand the cellular mechanisms leading to heart failure in Fog2MC mutants, we asked whether Fog2 inactivation resulted in increased cardiomyocyte apoptosis. Cardiomyocyte apoptosis was significantly increased in Fog2MC hearts compared with either Fog2fl/+Myh6-Cre and Fog2fl/–NTg controls, as determined by both TUNEL and activated caspase-3 staining (Figure 4, G–I). Consistent with increased cardiomyocyte apoptosis, fibrotic replacement of apoptotic myocardial tissues resulted in increased cardiac fibrosis in Fog2MC hearts, as demonstrated by both trichrome and collagen III staining (Supplemental Figure 7, C–F).
Fog2 is required for normal myocardial perfusion and coronary angiogenesis. Based on defective coronary vasculogenesis observed in fetal Fog2NK hearts, we hypothesized that abnormal coronary vasculature may contribute to the development of myocardial dysfunction and cardiomyocyte apoptosis in Fog2MC hearts. In vivo injection of a latex polymer (Microfil) revealed decreased coronary vasculature in Fog2MC hearts compared with control hearts (Figure 5, A–F). This was confirmed by tail vein injection of Bs-1 lectin, which stained endothelial cells that received effective blood flow (Figure 5G). Decreased myocardial perfusion was further confirmed by measuring myocardial uptake of 99mTc-2-methoxy isobutyl isonitrile (MIBI), which is proportional to myocardial perfusion. MIBI uptake was significantly decreased in Fog2MC hearts (Figure 5H).
Defective myocardial perfusion and coronary vasculature in adult Fog2MC heart. (A–F) Cleared, Microfil-injected control and Fog2MC hearts. Microvascular filling by Microfil was markedly reduced in Fog2MC hearts compared with control. (G) Perfusion labeling of endothelial cells by injected Bs-1 lectin. Myocardial labeling was reduced in Fog2MC. (H) Assessment of myocardial perfusion by myocardial MIBI uptake. Uptake is expressed as a fraction of the injected dose, normalized to heart weight. (I and J) Representative figure of hypoxia in Fog2MC myocardium. Hypoxia was detected as immunoreactivity to hypoxyprobe (hypoxy). Staining was greater in Fog2MC myocardium, including cardiomyocytes (arrowheads). Boxed regions are magnified in center panels. Transmission” indicates phase-contrast images. (K) Decreased capillary density in Fog2MC myocardium. PECAM+ vessels in histological sections were quantitated. (L) Decreased smooth muscle–positive vessels in Fog2MC myocardium. (M) Increased endothelial cell apoptosis in Fog2MC myocardium. The boxed apoptotic endothelial cell is magnified (right panel). Apoptosis was quantitated by TUNEL staining of PECAM+ coronary endothelial cells (white arrow). For quantitation in H and K–M, n = 3–4 for each group. *P < 0.05. Scale bar: 500 μm (A, B, D, and E); 250 μm (C and F); 50 μm (G and I–M).
Decreased myocardial perfusion and increased cardiomyocyte apoptosis in Fog2MC hearts suggested the possibility that the mutant myocardium was ischemic. To test this hypothesis, we injected the compound pimonidazole hydrochloride (hypoxyprobe), which forms protein adducts under hypoxic conditions. These protein adducts were visualized with specific antibodies. Hypoxyprobe immunoreactivity was substantially increased in Fog2MC hearts compared with littermate Fog2fl/–NTg controls (Figure 5, I and J). Both cardiomyocytes and interstitial cells were stained. These data indicated that diminished perfusion in Fog2MC hearts resulted in tissue hypoxia, which likely contributed to elevated cardiomyocyte apoptosis.
Consistent with these findings, the density of PECAM+ vessels, predominantly capillaries, was significantly diminished in Fog2MC hearts compared with Fog2fl/+Myh6-Cre and Fog2fl/–NTg control hearts (204.1 ± 25.6 vs. 251.2 ± 13.0 and 255.0 ± 10.5 vessels/×40 field, respectively; P < 0.05; Figure 5K). The decreased density of endothelial cells was associated with increased endothelial cell apoptosis in Fog2MC hearts, as demonstrated by quantitative analysis of TUNEL and activated caspase-3–stained sections (Figure 5M and Supplemental Figure 7J). SMA staining revealed that Fog2MC hearts also had lower coronary arteriole density compared with Fog2fl/+Myh6-Cre and Fog2fl/–NTg control hearts (11.2 ± 3.5 vs. 21.1 ± 4.5 and 18.9 ± 3.8 vessels/×20 field, respectively; P < 0.05; Figure 5L). These results indicate that myocardial Fog2 expression is essential for maintenance of coronary vasculature.
Induced inactivation of Fog2 in postnatal myocardium leads to cardiac failure. Fog2 is expressed in both fetal and adult heart (Figure 1 and Supplemental Figure 8, A–D). While the Fog2MC phenotype suggested that Fog2 continues to have an important role in the adult heart, an alternate possibility was that the adult Fog2MC phenotype is an adult manifestation of a developmental phenotype. Therefore, to directly address the requirement of Fog2 in the adult heart, we developed an inducible, cardiomyocyte-restricted Fog2 inactivation model. We generated a Cre transgene, named TNT-iCre, by co-integrating 2 constructs, one containing the cardiac-specific troponin T promoter driving the reverse tet activator protein (rtTA) and the other containing a tet activator protein-dependent promoter driving Cre (Figure 6A). Administration of doxycycline (Dox) upregulates Cre expression in cardiomyocytes, resulting in Cre-mediated recombination of floxed targets. Dox-treated TNT-iCre strongly recombined the Rosa26fsLz reporter in cardiomyocytes (Supplemental Figure 8, E–G).
Postnatal inactivation of Fog2 caused cardiac failure. (A) Fog2 inactivation strategy in adult mice. cTnT, cardiac troponin T promoter; ex8, Fog2 exon 8; Fog2Δ, Cre-inactivated Fog2 allele; rtTA, reverse tet activator protein; TRE, tet activator protein response element. Red triangles indicate loxP sites. (B) Decreased Fog2 expression in Dox-treated Fog2iCre heart apexes compared with controls. (C) Decreased ventricular function in Fog2iCre mice after 4 weeks of Dox treatment. (D and E) Time course of Fog2 inactivation and ventricular systolic function during Dox treatment. Fog2 expression was determined by qRT-PCR (n = 4 per time point). (F) Electron microscopy showed that sarcomere and mitochondrial structures were well preserved in Fog2iCre–Dox mutant hearts. Scale bar: 500 nm. (G and H) PECAM (red) staining showed decreased density of coronary blood vessels in Fog2iCre–Dox hearts compared with those of controls. n = 3 per group. Scale bar: 50 μm. (I and J) Quantitation of TUNEL+ coronary endothelial cells (ECs) and cardiomyocytes. Apoptosis increased in Fog2iCre–Dox hearts compared with those of Fog2fl/fl littermate controls. n = 3 per group. (K) Trichrome staining demonstrating increased fibrotic tissues in Fog2iCre–Dox hearts. Scale bar: 50 μm. *P < 0.05.
To inducibly inactivate Fog2 in adult cardiomyocytes, we generated Fog2fl/flTNT-iCre mice (designated Fog2iCre). Mice were treated with standard or Dox-containing drinking water from 4 to 8 weeks of age. Mice then underwent echocardiography and pathological examination. In the absence of Cre, Dox treatment of Fog2fl/fl mice did not significantly affect Fog2 expression. In Fog2iCre hearts not treated with Dox (Fog2iCre–Ctrl), Fog2 expression was also not significantly changed, indicating minimal recombination in the absence of Dox. In contrast, Fog2 expression in Fog2iCre hearts treated with Dox (Fog2iCre–Dox) was significantly decreased, indicating efficient Dox-induced inactivation of Fog2 (Figure 6B).
Cardiomyocyte-restricted Fog2 inactivation induced in adulthood impaired cardiac systolic function, as indicated by significantly diminished fractional shortening in Fog2iCre–Dox hearts compared with Fog2iCre–Ctrl hearts (P < 0.001; Table 2 and Figure 6C). This effect was not due to Dox treatment itself, as Dox did not affect systolic function of Fog2fl/flNTg hearts (Figure 6C). To further confirm the relationship between reduced Fog2 expression and heart function, we monitored Fog2iCre and control mice after Dox treatment for 0, 4, 8, 14, and 21 days. Both Fog2 expression and fractional shortening progressively decreased in Fog2iCre mice over the course of the study, with decrease in Fog2 expression appearing to precede cardiac dysfunction by approximately 1 week (Figure 6, D and E; e.g., compare days 14 and 21). Consistent with our findings in Fog2MC mice, cardiac dysfunction in Fog2iCre–Dox mice was not associated with significant changes in cardiomyocyte sarcomeric or mitochondrial structure (Figure 6F).
As in Fog2MC mice, PECAM staining of Fog2iCre–Dox tissue demonstrated diminished coronary vascular density (Figure 6, G and H). Consistent with this, Microfil injection suggested that the coronary vasculature was less extensive in Fog2iCre–Dox mice compared with Fog2iCre–Ctrl mice (data not shown). Decreased coronary vascular density was associated with increased apoptosis of Fog2iCre–Dox endothelial cells, as demonstrated by both TUNEL and activated caspase-3 staining (Figure 6I and data not shown). Cardiomyocyte apoptosis likewise increased in Fog2iCre–Dox hearts (Figure 6J), suggesting that increased cardiomyocyte loss contributed to impairment of systolic function. In keeping with increased cardiomyocyte loss, more fibrosis was also noticed in Fog2iCre–Dox hearts compared with Fog2iCre–Ctrl hearts (Figure 6K). Collectively, these data demonstrated that Fog2 expression in adult cardiomyocytes is necessary to maintain heart function and coronary vasculature.
Block of FOG2-GATA4 interaction resulted in postnatal heart failure. Given the requirement of GATA4 for normal function of the adult heart (7, 8) and the requirement of GATA4-FOG interaction for heart development (13), we hypothesized that FOG2 action in the adult heart is mediated by interaction with GATA4. To test this hypothesis, we used a conditional complementation approach. We complemented Gata4ki, a missense Gata4 allele encoding a GATA4[V217G] protein specifically deficient in FOG2 interaction (13), with Gata4fl, a floxed Gata4 allele. In embryo heart, Cre-recombined Gata4fl produced a functionally inactivate, truncated GATA4 protein (2), while in adult heart, it did not express detectable protein (7). Thus, in Gata4fl/kiCre+ embryos, GATA4[V217G] is the sole full-length GATA4 protein within the Cre recombination domain.
We generated Gata4fl/kiNkx2-5Cre/+ (Gata4ki–NK) and Gata4fl/kiMyh6-Cre (Gata4ki–MC) embryos. Nkx2-5Cre/+ and Myh6-Cre efficiently inactivated Gata4fl by E9.5 and E12.5, respectively (6). Early loss of GATA4-FOG2 interaction in Gata4ki–NK embryos resulted in edema and peripheral hemorrhage, consistent with heart failure (Supplemental Figure 9A). Morphologically, Gata4ki–NK embryos developed a spectrum of cardiac defects similar to those seen in Fog2NK embryos: thin compact myocardium, AV cushion defect, VSD, and impaired coronary vascular development (Supplemental Figure 9, B–D). Importantly, Gata4fl/+Nkx2-5Cre/+ embryos developed normally, suggesting that the phenotype of Gata4ki–NK embryos is not due to Gata4 or Nkx2-5 haploinsufficiency. These data strongly indicated that FOG2-GATA4 interaction in cardiomyocytes is critical for cardiac morphogenesis and coronary vascular development.
As in Fog2MC mice, later ablation of FOG2-GATA4 interaction in Gata4ki–MC embryos was compatible with normal survival to term but caused severely diminished systolic function at 8–14 weeks as shown by echocardiography and PET imaging (Figure 7A, Table 2, and Supplemental Videos 3 and 4). This was not due to haploinsufficiency for either GATA4 or GATA4-FOG2 interaction, as controls with Gata4fl/+Myh6-Cre and Gata4fl/kiNTg genotypes had normal systolic function (Figure 7A and Table 2). Histological analysis showed ventricular dilatation and fibrosis (Supplemental Figure 10, E and F). Consistent with heart failure, Anf and Bnp mRNAs were significantly upregulated (Supplemental Figure 9G). Serca2 expression was unchanged (Supplemental Figure 9, G and H). Fog2 expression was also unchanged, indicating that the Gata4ki–MC phenotype was not secondary to epistasis between Gata4 and Fog2.
Heart failure due to disruption of FOG2-GATA4 interaction. (A) Severely decreased heart function in Gata4ki–MC mice compared with Gata4fl/+Myh6-Cre or Gata4fl/kiNTg. (B and C) Quantitation of TUNEL+ cardiomyocytes and coronary endothelial cells showed increased apoptosis in Gata4ki–MC hearts compared with those of controls (n = 3 per group). (D) Injected Microfil demonstrated reduced coronary microvasculature in Gata4ki–MC hearts compared with littermate control (Gata4+/flMyh6-Cre). Scale bar: 1 mm (top panels); 200 μm (bottom panels). (E) PECAM staining revealed decreased capillary density in Gata4ki–MC hearts compared with controls (n = 3). (F) SMA staining revealed decreased arteriole density in Gata4ki–MC hearts compared with controls (n = 3). (G) Fibronectin and collagen III (Col III) staining revealed significantly increased fibrosis in Gata4ki–MC hearts compared with controls (n = 3). Scale bar: 100 μm. *P < 0.05.
Cardiomyocyte and endothelial cell apoptosis were significantly increased in Gata4ki–MC mutants compared with controls (Figure 7, B and C). Injection of Microfil revealed that the coronary vasculature of Gata4ki–MC hearts was dramatically diminished, suggesting impaired myocardial perfusion in Gata4ki–MC hearts (Figure 7D). Consistent with these data and the Fog2MC phenotype, Gata4ki–MC hearts had reduced coronary capillary and arteriolar density compared with control hearts, as determined by quantitative analysis of PECAM- and SMA-stained sections (P < 0.05; Figure 7, E and F). Cardiac fibrosis was also increased in Gata4ki–MC (Figure 7G). Together, these data indicate that FOG2-GATA4 interaction is critical for postnatal cardiac function and maintenance of coronary vasculature.
Regulation of a proangiogenic gene program by Fog2. To pursue the molecular mediators of Fog2 action in the postnatal heart, we used microarrays to compare gene expression in 6-week-old Fog2MC and control (Fog2fl/–NTg) heart ventricles (n = 3 per group). Six hundred and forty genes were differentially expressed (P < 0.005), with 248 downregulated and 392 upregulated. Among these genes were Fog2 itself and Anp and Bnp, findings that were supported by qRT-PCR (Figure 4, B and F). Additional intriguing dysregulated genes were cardiotrophin 1 (Ctf1; downregulated), the potassium voltage-gated channel, Isk-related family, member 1 (Kcne1, also known as MinK; upregulated), Ppara (downregulated), and the forkhead box C1 (Foxc1; upregulated). We measured these by qRT-PCR, and confirmed altered expression of all except Ppara. KCNE1 is an essential subunit of the cardiac slow delayed rectifier (IKs) channel, which contributes to cardiac repolarization. Although Kcne1 mRNA was dramatically upregulated, we did not observe arrhythmia or altered electrocardiographic parameters in Fog2MC mice (Supplemental Table 1 and data not shown). Altered expression of Ctf1 and Foxc1 may contribute directly to cardiomyocyte dysfunction or apoptosis in Fog2MC hearts (see Discussion).
Few angiogenesis-related genes were uncovered in our microarray screen. However, at a sample size of 3, our microarray screen lacked statistical power to detect many important biological changes. Therefore, we assembled a list of angiogenesis-related genes from the literature, and examined the microarray data for suggestive gene expression changes. We then directly measured expression of 17 angiogenic factors with suggestive microarray changes by qRT-PCR, comparing Fog2MC with controls with sample sizes increased to 8 per group. Expression of 14 of these angiogenesis-related genes was altered (Figure 8A). The proangiogenic genes Vegfa, Fgf2, Fgf9, Fgf12, and Fgf16 were significantly downregulated, while the angiogenesis inhibitors thrombospondin 1 (Thbs), tissue inhibitor of metalloproteinase 1 (Timp1), Timp2, heparanase (Hpse), and collagen, type IV, α 3 (Col4a3), collagen, type XV, α 1 (Col15a), and collagen, type XVIII, α 1 (Col18a) were upregulated. Next, we asked whether FOG2 acts through GATA4 to regulate these genes. We compared expression of these same genes between Gata4ki–MC and control hearts (n = 7–9 per group). Expression of nearly all of these genes was also disrupted in Gata4ki–MC, and the direction of change was concordant with that observed in Fog2MC (Figure 8B). These data indicate that a FOG2-GATA4 complex coordinates expression of a set of pro- and antiangiogenesis genes, acting to promote an overall proangiogenic gene expression profile.
Fog2 regulation of angiogenesis. (A) Relative expression of genes encoding secreted angiogenesis-related factors in Fog2MC heart apex compared with controls. Gene expression was measured by qRT-PCR. n = 8 per group. Thbs, thrombospondin 1; Timp1, tissue inhibitor of metalloproteinase 1; Hpse, heparanase; Col4a3, collagen, type IV, α 3; Col15a, collagen, type XV, α 1; Col18a, collagen, type XVIII, α 1; Ctgf, connective tissue growth factor; Fn1, fibronectin 1. (B) Relative expression of genes encoding secreted angiogenesis-related factors in Gata4ki–MC heart apex (n = 7) compared with controls (n = 9). (C) Depletion of Fog2 in cultured mouse neonatal cardiomyocytes. Fog2fl/fl cardiomyocytes treated with Cre adenovirus showed reduced FOG2 immunoreactivity (white arrowheads), while FOG2 was readily detected after treatment with control LacZ adenovirus (yellow arrowheads). (D) Relative expression of Fog2, as assessed by qRT-PCR. Cre strongly reduced Fog2 expression compared with LacZ. (E) Representative images of tubule formation by HUVECs plated on matrigel and cocultured with Cre or LacZ virus–treated Fog2fl/fl cardiomyocytes. (F) Fog2fl/fl cardiomyocytes stimulated less HUVEC tubule formation after Cre treatment than after LacZ treatment. n = 3. (G) BrdU staining of PECAM+ HUVECs cocultured with cardiomyocytes (sarcomeric α actinin [Actn2]). Scale bar: 20 μm. (H) The percentage of BrdU+ cells in HUVECs cocultured with Fog2fl/fl cardiomyocytes that have been treated with Cre or LacZ adenovirus (n = 5). *P < 0.05.
Coordination of cardiac growth and angiogenesis is in part mediated by the secretion of angiogenic growth factors from myocytes (28). The abnormal expression profile of angiogenesis-related genes suggested that _Fog2_-deficient cardiomyocytes were defective in their ability to support endothelial growth and vessel formation. To further investigate the functional role of cardiomyocyte FOG2 in regulating angiogenesis, we used an in vitro cardiomyocyte–endothelial cell coculture system. Primary neonatal Fog2fl/fl cardiomyocytes were cultured on matrigel and treated with adenovirus expressing either Cre or LacZ (negative control). Cre adenovirus efficiently reduced Fog2 expression (Figure 8, C and D). Next, we plated HUVECs on the cardiomyocytes and assessed tubule formation. The quality and quantity of tubule formation was significantly decreased by cardiomyocyte inactivation of Fog2 (Figure 8, E and F), suggesting that Fog2 regulates a proangiogenic gene program in cardiomyocytes that promotes capillary-like structure formation. In addition, we asked whether cardiomyocyte Fog2 regulates endothelial cell proliferation. Cardiomyocyte Fog2 inactivation decreased HUVEC BrdU incorporation (Figure 8, G and H), indicating that Fog2 regulates the expression of cardiomyocyte-secreted factors that stimulate endothelial cell proliferation.
In summary, we performed temporal and spatial inactivation of Fog2 in embryos and found early, but not late, requirement of FOG2 expression and FOG2-GATA4 interaction for cardiac morphogenesis and coronary vasculogenesis. In addition, maintenance of postnatal heart function also required FOG2 and its interaction with GATA4. Fog2 regulates a subset of angiogenesis-related genes, and loss of Fog2 caused decreased coronary vasculature and tissue hypoxia, which subsequently resulted in cardiomyocyte cell death and heart failure.