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Circulation Research. 2003;92:1305-1313
Published online before print May 15, 2003, doi: 10.1161/01.RES.0000077045.84609.9F
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(Circulation Research. 2003;92:1305.)
© 2003 American Heart Association, Inc.

Molecular Medicine

NFATc3 and NFATc4 Are Required for Cardiac Development and Mitochondrial Function

Paul B. Bushdid, Hanna Osinska, Ronald R. Waclaw, Jeffery D. Molkentin, Katherine E. Yutzey

From the Divisions of Molecular Cardiovascular Biology (P.B.B., H.O., J.D.M., K.E.Y.) and Developmental Biology (R.R.W.), Children’s Hospital Medical Center Cincinnati, Cincinnati, Ohio.

Correspondence to Katherine E. Yutzey, Children’s Hospital Medical Center Cincinnati, ML 7020, 3333 Burnet Ave, Cincinnati, OH 45229. E-mail yutzey{at}chmcc.org


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Activation of the nuclear factor of activated T-cell (NFAT) family of transcription factors is associated with changes in gene expression and myocyte function in adult cardiac and skeletal muscle. However, the role of NFATs in normal embryonic heart development is not well characterized. In this report, the function of NFATc3 and NFATc4 in embryonic heart development was examined in mice with targeted disruption of both nfatc3 and nfatc4 genes. The nfatc3-/-nfatc4-/- mice demonstrate embryonic lethality after embryonic day 10.5 and have thin ventricles, pericardial effusion, and a reduction in ventricular myocyte proliferation. Cardiac mitochondria are swollen with abnormal cristae, indicative of metabolic failure, but hallmarks of apoptosis are not evident. Furthermore, enzymatic activity of complex II and IV of the respiratory chain and mitochondrial oxidative activity are reduced in nfatc3-/-nfatc4-/- cardiomyocytes. Cardiac-specific expression of constitutively active NFATc4 in nfatc3-/-nfatc4-/- embryos prolongs embryonic viability to embryonic day 12 and preserves ventricular myocyte proliferation, compact zone density, and trabecular formation. The rescued embryos also maintain cardiac mitochondrial ultrastructure and complex II enzyme activity. Together, these data support the hypothesis that loss of NFAT activity in the heart results in a deficiency in mitochondrial energy metabolism required for cardiac morphogenesis and function.


Key Words: nuclear factors of activated T cells • heart development • mitochondria


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The nuclear factor of activated T-cell (NFAT) family of transcription factors contributes to the development of several organ systems, including the immune system, vasculature, nervous system, and heart.1,2 Five NFAT genes, nfatc1–4 and nfat5, have been identified, each with distinct temporally and spatially regulated expression patterns.3 Mutation of NFAT genes affects the development of endothelial cells, skeletal myoblasts, chondroblasts, keratinocytes, and adipocytes. The cardiovascular system, the first functional organ system in the developing embryo, is dependent on NFAT activity for development and function during embryogenesis and for adult adaptation to cardiac stress.2,4 Mutation of nfatc1 results in cardiac valve and septal defects, whereas overexpression of NFATc4 induces cardiac hypertrophy in adult mice.2,4 In skeletal muscle, NFATc3 activity is critical for the generation of primary myofibers, whereas NFATc2 regulates skeletal muscle growth.5 Furthermore, combinatorial disruption of nfatc3 and nfatc4 results in embryonic lethality around embryonic day 11 (E11) that is due to cardiovascular defects with abnormal vascular patterning.6 These data confirm the importance of NFAT activity in cardiac and skeletal muscle development; however, the precise role(s) of NFATc3 and NFATc4 in heart formation remains unclear.

The NFATc transcription factors contain a conserved calcineurin (CNA) binding domain and a Rel-homology DNA binding domain.1,3 NFATs are activated by the protein phosphatase, CNA, in response to increased intracellular Ca2+. Dephosphorylated NFATs translocate to the nucleus, bind DNA, and regulate gene expression.7 Changes in ß-myosin heavy chain (ß-MyHC), atrial natriuretic factor, brain natriuretic factor, sarco/endoplasmic reticulum Ca2+-ATPase 2, and {alpha}-myosin heavy chain expression ({alpha}-MyHC) occur with NFAT activation in the adult hypertrophic response.7–9 Moreover, NFATc3 and NFATc4 act in conjunction with other transcription factors, such as GATA, myocyte enhancer factor 2, and serum response factor, to effect changes in gene expression during hypertrophy and fiber-type switching in cardiac and skeletal muscle.9–11 Therefore, in muscle, NFAT activation in response to elevated Ca2+ regulates gene expression during development, maturation, and adaptation to external stimuli.

Adult cardiac hypertrophy is associated with activation of NFAT, alteration of myofiber type, and depletion of energy stores.5,12 The changes in gene expression associated with adult cardiac hypertrophy mirror the fetal ventricular gene profile and are considered a reversion to embryonic regulatory pathways.4,5,8 The fetal ventricular gene profile established at E10.5 to E11.5 of mouse development includes chamber restriction of contractile protein, Ca2+ handling, and metabolic gene expression.13 In rat embryos, this stage of development is also characterized by cardiac mitochondrial ultrastructural maturation and increased dependence on oxidative phosphorylation for energy production.14–16 This midgestation transition from glycolytic to oxidative metabolism in the developing heart is reminiscent of the metabolic switch observed in the fast- to slow-twitch skeletal myofiber differentiation associated with NFAT activity.10,11 Furthermore, recent data have shown that NFATs either directly or indirectly regulate the expression of metabolic proteins essential in muscle energy production, such as adenyl succinate synthase 1 and carnitine palmitoyltransferase 1.17,18 Together, these data are consistent with a regulatory role for NFATs in skeletal and cardiac muscle contractile protein gene expression and bioenergetics.

The role of NFATc3 and NFATc4 in cardiac muscle development was examined in nfatc3-/-nfatc4-/- embryos. The combinatorial disruption of nfatc3 and nfatc4 results in abnormal heart morphogenesis by E10.25 and embryonic lethality at {approx}E11. Cellular, molecular, and metabolic analyses of nfatc3-/-nfatc4-/- embryos at E10.5 have demonstrated that cardiomyocyte proliferation is decreased and that cardiac mitochondrial architecture and function are compromised. Evidence for a primary defect in myocardial metabolism is provided by reduced mitochondrial oxidative activity in cultured cardiomyocytes isolated from E9.5 nfatc3-/- nfatc4-/- embryos before any morphological defects. The requirement for NFAT activity specifically in the heart was examined using cardiac-specific transgenic expression of NFATc4.9 Restoration of NFAT activity, specifically in the cardiomyocytes of nfatc3-/-nfatc4-/- embryos by transgenesis results in prolongation of embryonic viability and maintenance of mitochondrial morphology and function. These observations demonstrate the requirement for NFATc3/NFATc4 activity in the maintenance of cardiomyocyte function and bioenergetics during development.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
A detailed Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Morphological Analysis of nfatc3/nfatc4 Mutant Embryos Reveals Defective Cardiac Development
Nfatc3 and nfatc4 mutant mice were bred together to investigate whether the combined loss of NFATc3 and NFATc4 affects myocardial development. Nfatc3 and nfatc4 are both expressed in developing cardiomyocytes; however, singular inactivation of either does not affect cardiac development.6,19,20 Embryos isolated from nfatc3+/-nfatc4-/- intercrosses were apparently normal at E9.5 and were indistinguishable from one another regardless of genotype. At E10.5, nfatc3-/-nfatc4-/- embryos exhibited defects in cardiac development, which were exemplified by dilated thin translucent hearts, pericardial effusion, and anemia (Figure 1C). Despite a mild generalized developmental delay, the heads, tails, and limb buds were well developed in the E10.5 nfatc3-/-nfatc4-/- embryos, and diffuse hemorrhaging was not apparent. By E11.5, nfatc3-/-nfatc4-/- embryos were either necrotic or resorbed, and no viable nfatc3-/-nfatc4-/- embryos were obtained at later time points (see online Table 1, available at http://www.circresaha.org). These observations suggest that the death of nfatc3-/-nfatc4-/- embryos soon after E10.5 is likely due to cardiovascular failure.



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  		Figure 1. Morphological analysis of nfatc3-/-nfatc4-/- embryos. Litters from nfatc3+/-nfatc4-/- crosses were examined at E10.5 for malformations in heart development. A and B, The gross morphology of nfatc3+/+nfatc4-/- embryos at E10.5 is normal. Primitive ventricles have normal trabeculation and thickening of the compact zone of the primitive ventricle (n=6). C and D, Thinning of the nfatc3-/-nfatc4-/- heart is evidenced by its translucent nature (arrow) and reduction in trabeculation and thin compact zone (n=8). Head (asterisks) and limbs (arrowheads) appear normal. E, Quantification of compact zone cell number (area between the 2 arrowheads in panels B and D) of littermates reveals a 50% reduction in the number of cell layers in the compact zones of the developing ventricular myocardium of nfatc3-/-nfatc4-/- embryos (*P≤0.001). Bar=10 µm.

Cardiac morphological defects associated with the loss of NFATc3/NFATc4 activation were examined in histological sections of E10.5 nfatc3+/+nfatc4-/- and nfatc3-/-nfatc4-/- embryos. Nfatc3+/+nfatc4-/- ventricular myocardium demonstrated normal trabeculation and thickening of the compact zone (Figure 1B). In contrast, nfatc3-/-nfatc4-/- ventricular trabeculae were thinner and fewer in number, and the compact zone was composed of fewer cells (Figure 1D). Quantification of cell layers in the compact zone revealed >50% reduction (P≤0.001) in the nfatc3-/-nfatc4-/- ventricular compact zone relative to the nfatc3+/+nfatc4-/- and nfatc3+/- nfatc4-/- littermates (Figure 1E). Histological sections of nfatc3+/+nfatc4+/+, nfatc3-/-nfatc4+/+, and nfatc3+/-nfatc4-/- embryos were equivalent to nfatc3+/+nfatc4-/- embryos (data not shown). Therefore, there is a specific decrease in cell number in the nfatc3-/-nfatc4-/- ventricular myocardium relative to same-stage embryos with at least one nfatc3 or nfatc4 allele.

Myocardial Proliferation Is Decreased in nfatc3/nfatc4 Mutant Embryos
Possible causes for the thin ventricular myocardium noted in nfatc3-/-nfatc4-/- embryos include defective differentiation of cardiomyocytes, lack of proliferation, and increased cell death. E9.5 to E10.5 nfatc3-/-nfatc4-/- cardiomyocytes beat and express ß-MyHC, suggesting that the cardiac defects observed are not due to a defect in primary differentiation (data not shown). To address the role of proliferation in myocardial development in nfatc3-/-nfatc4-/- embryos, bromodeoxyuridine (BrdU) incorporation was used as an indicator of DNA synthesis in cells in S phase. Pregnant females carrying mixed litters of nfatc3/nfatc4 mutant embryos were injected on E10.5 with the thymidine analogue, BrdU. Proliferation indices were calculated as the number of BrdU+ cells per total number of cells in histological sections immunostained for BrdU (Figure 2A). Immunohistochemistry for BrdU incorporation revealed >50% reduction in proliferation in nfatc3-/-nfatc4-/- myocardium compared with embryos with at least one nfatc3/nfatc4 allele (P≤0.001). No significant differences in proliferation were noted between E10.5 nfatc3+/+nfatc4+/+, nfatc3+/+nfatc4-/-, nfatc3-/-nfatc4+/+, and nfatc3+/-nfatc4-/- ventricular myocardium. Furthermore, comparable proliferation of pharyngeal arch mesenchyme was noted between nfatc3-/-nfatc4-/- and nfatc3+/+nfatc4+/+ embryos. These data demonstrate a significant decrease specifically in ventricular cardiomyocyte proliferation on loss of NFATc3 and NFATc4.



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  		Figure 2. Analysis of proliferation and cell death in E10.5 nfatc3-/-nfatc4-/- hearts. A, Quantification of BrdU incorporation demonstrates a 50% reduction in myocardial proliferation in nfatc3-/-nfatc4-/- embryos (n=4; *P≤0.001). No difference in BrdU incorporation was observed in pharyngeal arch mesenchyme. B and C, Ultrastructural analysis (original magnification x10 000) using TEM revealed normal nuclei (N) for both genotypes. Pyknotic nuclei, a hallmark of apoptosis, are not observed. Mitochondria (arrowheads) are normal in nfatc3+/+nfatc4-/- embryos but appear swollen with decreased crista formation in nfatc3-/-nfatc4-/- cardiomyocytes (n=4). D and E, Nile blue staining for cell death is identical in nfatc3+/+nfatc4-/- (D, n=9) and nfatc3-/-nfatc4-/- (E, n=7) embryos. Nile blue was detected as a dark blue stain in the limb buds, somites, and branchial arches (arrowheads) of both genotypes but was not observed in the developing ventricles (asterisks).

The possibility of an increase in cardiomyocyte death on the loss of nfatc3/nfatc4 was examined by transmission electron microscopy (TEM) and vital dye staining at E10.5. Nuclei of nfatc3-/-nfatc4-/- hearts were examined for hallmarks of apoptosis using TEM (Figures 2B and 2C). Apoptotic cells exhibit pyknotic nuclei, membrane blebbing, and apoptotic bodies within the cytoplasm.21 No increase in morphological evidence of apoptosis was observed in TEMs of nfatc3-/-nfatc4-/- hearts relative to nfatc3+/+nfatc4-/- hearts. In each case, <0.5% of the cells exhibited apoptosis consistent with normal levels in heart development. Ultrastructural analysis of nfatc3-/-nfatc4-/- cardiomyocytes reveals nuclei with intact membranes and distinct nucleoli, characteristic of normal healthy bioactive cells.21 To confirm the TEM results indicating no increase in cell death, E10.5 embryos were stained with Nile blue, a vital dye that stains dead or dying cells (Figures 2D and 2E). Nile blue staining is typically seen in areas of active apoptosis, such as limb buds, branchial arches, and somites.22 Normal patterns of cell death were revealed by Nile blue staining in nfatc3+/+nfatc4-/-, nfatc3+/-nfatc4-/-, and nfatc3-/-nfatc4-/- embryos. Notably, no increase in vital dye staining was observed in the hearts of E10.5 nfatc3-/-nfatc4-/- embryos. Together, these analyses demonstrate that the thin ventricles of nfatc3-/-nfatc4-/- embryos result from decreased proliferation and not from lack of cardiomyocyte differentiation or increased cell death.

Cardiomyocytes Lacking NFATc3 and NFATc4 Have Mitochondrial Defects
TEM was further used to assess cellular ultrastructure to determine whether the cardiac contractile apparatus and/or organelles were affected by the loss of NFATc3/NFATc4 activity. Analysis of nfatc3/nfatc4 mutant littermates at E10.5 revealed defects in the subcellular architecture of nfatc3-/- nfatc4-/- cardiomyocytes (n=4). Nfatc3+/+nfatc4-/- and nfatc3-/-nfatc4-/- embryos had blood cells in the heart, intact endocardium, and normal-appearing sarcomeric organization (Figures 3A and 3B). Notably, nfatc3-/-nfatc4-/- ventricular myocardium contained enlarged disorganized mitochondria with malformed cristae, whereas mitochondria in nfatc3-/- nfatc4-/- noncardiac tissue (data not shown) and nfatc3+/+ nfatc4-/- embryos were normal in appearance (Figures 3C and 3D, Figures 2B and 2C). The abnormal mitochondria were grossly swollen and exhibited multiple defects in cristae formation, including few or absent cristae, dilated tubules, curvilinear malformation, or onion-like swirls. Similar mitochondrial abnormalities were observed mouse embryos lacking Pex5, an oxidative metabolism gene essential for lipid transport and ß-oxidation.23 The aberrant mitochondria could feasibly be associated with apoptosis, but no hallmarks of apoptosis21 were observed (Figures 2B and 2C). The similarity between mitochondrial abnormalities observed in Pex5 mutant embryos with definitive metabolic defects and in nfatc3-/-nfatc4-/- cardiomyocytes is suggestive of metabolic failure in hearts lacking NFATc3 and NFATc4.



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  		Figure 3. Mitochondrial ultrastructure and respiratory activity is reduced in E10.5 nfatc3-/-nfatc4-/- embryos. A and B, Normal sarcomeres (S) are evident by TEM in nfatc3+/+nfatc4-/- (A) and nfatc3-/-nfatc4-/- (B) cardiomyocytes (n=4). C and D, Ultrastructural analysis of the mitochondria (M) in E10.5 nfatc3-/-nfatc4-/- embryos (D), however, reveals grossly misshapen mitochondria with few cristae relative tonfatc3+/+nfatc4-/- embryos (C) at the same magnification. E and F, Mitochondrial function as measured by CoxC activity is detected as a dark precipitate in CoxC-positive mitochondria. Cardiomyocytes from nfatc3+/+nfatc4-/- embryos (E) demonstrate >90% CoxC-positive mitochondria (arrow), whereas >60% CoxC-negative mitochondria (arrowhead) are observed in nfatc3-/-nfatc4-/- cardiomyocytes (n=2). G and H, SDH activity, as observed by nitro blue tetrazolium precipitation, is reduced in the hearts of nfatc3-/-nfatc4-/- embryos (H), including the left ventricle (LV, n=5).

Swollen mitochondria with crista malformations associated with defective respiration are observed in mouse heart cells exposed to ethanol toxicity or cardiomyopathy related to diabetes.24,25 Therefore, the mitochondrial defects in nfatc3-/- nfatc4-/- embryos could be due to decreased respiratory function. Two components of the respiratory chain, cytochrome c oxidase (CoxC) and succinate dehydrogenase (SDH), were analyzed for enzymatic activity as indicators of mitochondrial function in nfatc3/nfatc4 mutant embryos.24–27 CoxC activity was detected in the cristae of virtually all the mitochondria in nfatc3+/+nfatc4-/- E10.5 hearts (Figure 3E). However, examination of nfatc3-/-nfatc4-/- embryos revealed loss of CoxC activity in the swollen malformed mitochondria (Figure 3F). CoxC activity remains in the few normal mitochondria present in the nfatc3-/-nfatc4-/- embryos, demonstrating a reduction in metabolic function and not complete loss. Analysis of SDH enzyme function in E10.5 whole hearts revealed a marked reduction in respiratory activity in nfatc3-/-nfatc4-/- hearts compared with nfatc3+/+nfatc4-/- littermate hearts (Figures 3G and 3H). However, sdh RNA levels were not significantly altered by the mutation of NFAT, as detected by real-time quantitative polymerase chain reaction (see online Figure 1A, available at http://www.circresaha.org). In addition, expression analysis of a variety of candidate genes involved in metabolism or mitochondrial biogenesis showed that the vast majority were not significantly affected by the loss of NFATc3 and NFATc4 (see online Table 2, available at http://www. circresaha.org). However, significant decreases in gene expression were observed in heart–fatty acid binding protein and pyruvate carboxylase, genes important for lipid oxidation and the Krebs cycle, respectively28,29 (see online Figure 1B). Together, these data indicate reduced cardiac metabolic function with associated defects in mitochondrial architecture in embryos lacking NFATc3 and NFATc4 activity.

Mitochondrial respiratory activity was further analyzed in cultures of E9.5 embryonic heart cells (Figure 4). NFAT mutant hearts are morphologically indistinguishable from wild-type hearts at E9.5, 1.5 to 2 days before lethality of nfatc3-/-nfatc4-/- embryos. Individually cultured E9.5 hearts from each genotype were stained with MitoTracker Red CM-H2XRos (Molecular Probes), a rhodamine derivative that fluoresces on oxidation and is sequestered by metabolically active mitochondria. Examination of cultures by confocal microscopy showed a marked decrease in mitochondrial fluorescence in nfatc3-/-nfatc4-/- cells compared with nfatc3+/+nfatc4-/- or nfatc3+/-nfatc4-/- cells (Figures 4A to 4C). Cultures that contained beating cardiomyocytes, ie, cells with normal morphology and strong TO-PRO3 nuclear DNA staining, were classified as viable and were subjected to further analysis. Cells undergoing necrosis or apoptosis would demonstrate nuclear breakdown or DNA condensation in the TO-PRO3 staining. Quantification of mitochondrial fluorescence from individual cells in viable cultures revealed a >50% decrease in respiratory activity in E9.5 cardiac cell cultures from nfatc3-/- nfatc4-/- embryos compared with nfatc3+/+nfatc4-/- or nfatc3+/-nfatc4-/- embryos (Figure 4D). These data indicate that the defect in metabolic activity is intrinsic to the cardiac cells isolated before any evidence of cardiovascular failure or embryonic lethality.



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  		Figure 4. Defects in mitochondrial function are intrinsic to the cardiomyocytes. E9.5 hearts were dissociated and grown on collagen-coated plates for 4 days. MitoTracker Red CM-H2Xros was used to assay for active mitochondrial oxidation. A through C, Representative confocal photomicrographs show the relative amounts of mitochondrial activity (red fluorescence) present in nfatc3+/+nfatc4-/- (A), nfatc3+/-nfatc4-/- (B), and nfatc3-/-nfatc4-/- (C) E9.5 cardiomyocyte cultures. Nuclei are fluorescent blue after staining with TO-PRO3. D, Quantification of mitochondrial fluorescence intensity revealed an {approx}50% decrease in mitochondrial oxidation in nfatc3-/-nfatc4-/- cardiomyocytes compared with nfatc3+/+nfatc4-/- cells (*P≤0.001). A minimum of 40 cells per culture and 4 cultures per genotype were assayed.

Major Blood Vessel Formation Occurs in nfatc3/nfatc4 Mutant Embryos
Targeted deletion of nfatc3 and nfatc4 has the potential to affect multiple organ systems throughout the embryo. It is possible that the cardiac defects could be a secondary effect of compromised vasculature.6 Therefore, Tie2LacZ transgenic mice were used to investigate the effect of nfatc3/nfatc4 deficiency on vascular development. Tie2LacZ mice express ß-galactosidase in vascular endothelial cells, making it possible to visualize vessel formation and patterning.30 The Tie2LacZ transgene was bred onto the nfatc3-/-nfatc4-/- background, and E10.5 embryos were examined for ß-galactosidase activity as an indicator of blood vessel formation. Examination of nfatc3+/+nfatc4-/-/Tie2LacZ and nfatc3-/-nfatc4-/-/Tie2LacZ littermates revealed formation of extensive head and body vasculature with either genotype (Figure 5). The major vessels in the head, including the internal carotid artery, were formed from the primitive vascular plexus in both genotypes, although they were somewhat rarefied in nfatc3-/-nfatc4-/- embryos (Figures 5A and 5B). Intersomitic vessels were clearly visible in E10.5 nfatc3-/-nfatc4-/-/Tie2LacZ embryos and were indistinguishable from vessels formed in nfatc3+/+nfatc4-/-/Tie2LacZ littermates (Figures 5C and 5D). Maturation of the vasculature involves the recruitment of smooth muscle cells to the wall of the developing large vessels, including the dorsal aorta. Immunohistochemistry for smooth muscle {alpha}-actin reveals the presence of smooth muscle cells in the wall of the dorsal aorta in nfatc3-/-nfatc4-/- embryos (Figures 5E and 5F). The number of smooth muscle cells present in the nfatc3-/-nfatc4-/- dorsal aorta is less than that observed in the nfatc3+/+nfatc4-/- dorsal aorta. However, no hemorrhaging is observed, suggesting sufficient vessel maturation in nfatc3-/-nfatc4-/- embryos to maintain vessel integrity under the hemodynamic conditions present.



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  		Figure 5. Vascular development appears normal in E10.5 nfatc3-/-nfatc4-/- embryos. A and B, ß-Galactosidase staining of endothelial cells shows comparable cephalic vessel formation in nfatc3+/+nfatc4-/- (A) and nfatc3-/-nfatc4-/- (B) embryos. Arrows indicate formation of one branch of the internal carotid artery (n=6). C and D, Formation of the intersomitic vessels (arrows) is indistinguishable between nfatc3+/+nfatc4-/- (C) and nfatc3-/-nfatc4-/- (D) embryos (n=6). E and F, Smooth muscle cells are observed in the wall of the dorsal aorta as brown cells (arrowheads) after immunohistochemical analysis for smooth muscle {alpha}-actin.

Cardiac-Specific Restoration of NFAT Activity Prolongs Embryonic Viability and Maintains Mitochondrial Ultrastructure
A genetic rescue of cardiac NFAT expression was initiated to examine the specific requirement for NFAT activity in heart development and to confirm cardiac deficiency as the primary cause of death in nfatc3-/-nfatc4-/- embryos. Transgenic mice expressing constitutively active human NFATc4 regulated by the {alpha}-MyHC promoter (NFATc4{Delta}317) were used to restore NFAT activity specifically in the heart.9 The NFATc4{Delta}317 protein lacks the N-terminal regulatory domain but contains the Rel-homology and transactivation domains, making it insensitive to CNA regulation and constitutively nuclear.9 Strong transgene expression was detected specifically in E8.5 to E10.5 developing hearts (Figures 6A to 6C). However, transgene expression was largely restricted to the atria by E11.5, with limited expression in the ventricles. The {alpha}-MyHC promoter transgene expression of NFATc4{Delta}317 is consistent with endogenous {alpha}-MyHC expression during these stages of development.13



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  		Figure 6. Cardiac-specific expression of constitutively active NFATc4 in the NFATc4{Delta}317 transgenic mouse line. In situ hybridization detects transgene expression through the developing heart between E8.5 and E10.5 (arrow). By E11.5, transgene expression is significantly diminished in the ventricles (V) but is retained in the atria (arrow).

The NFATc4{Delta}317 line was bred onto the nfatc3+/-nfatc4-/- background to restore NFAT activity specifically to the hearts of nfatc3-/-nfatc4-/- embryos. Expression of NFATc4{Delta}317 in nfatc3-/-nfatc4-/- embryonic hearts prolongs viability to {approx}E12 (Figure 7). Nfatc3-/-nfatc4-/-/NFATc4{Delta}317 embryos (n=14) were viable at E11.5 and were comparable to littermates with a functional nfatc3 allele, whereas nfatc3-/-nfatc4-/- embryos were necrotic by this stage (n=12, Figures 7A to 7C). Histological examination of the embryos revealed comparable cardiac development between nfatc3+/+nfatc4-/- and nfatc3-/-nfatc4-/-/NFATc4{Delta}317 embryos (Figures 7D and 7E). Compact zone density and trabecular formation were indistinguishable between the two genotypes. Furthermore, ventricular proliferation was sustained at 80% wild-type levels by NFATc4{Delta}317 expression (Figure 7G). No nfatc3-/-nfatc4-/-/NFATc4{Delta}317 embryos were alive at E12.5, when the transgene is no longer expressed in the ventricles. These data indicate that continued NFAT activity specifically in the heart prolongs embryonic survival in nfatc3-/- nfatc4-/- embryos.



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  		Figure 7. Cardiac-specific expression of NFATc4{Delta}317 prolongs viability of nfatc3-/- nfatc4-/- embryos. A and B, Nfatc3-/-nfatc4-/- embryos expressing cardiac-specific NFATc4{Delta}317 (B, n=14) were alive and indistinguishable from embryos expressing at least one nfatc3 or nfatc4 allele (A, n=45). C and F, Nontransgenic nfatc3-/-nfatc4-/- littermates were necrotic by E11.5 (n=12). D and E, Histological analysis reveals restoration of trabeculation in nfatc3-/-nfatc4-/-/NFATc4{Delta}317 ventricles (E) comparable to nfatc3+/+nfatc4-/- embryos (D). V indicates ventricle; A, atrium. G, Proliferation of the ventricular myocardium as measured by BrdU incorporation at E10.5 is restored in nfatc3-/-nfatc4-/-/NFATc4{Delta}317 embryos to >80% of wild-type levels (*P≤0.0001; {dagger}P≤0.001). Bar=10 µm.

Mitochondrial function and structure were restored by the expression of NFATc4{Delta}317 in nfatc3-/-nfatc4-/- cardiomyocytes. Analysis of SDH activity in E10.5 whole hearts revealed an {approx}30% reduction in respiratory activity in nfatc3-/-nfatc4-/- hearts that was restored to {approx}100% activity in nfatc3-/-nfatc4-/-/NFATc4{Delta}317 littermates (Figures 8A to 8C). Ultrastructural analysis of mitochondria from nfatc3-/-nfatc4-/- embryos revealed gross abnormalities in mitochondrial size and crista formation (Figures 2C, 3D, and 8 UpDownF). To quantify the abnormalities observed by TEM, mitochondria were categorized as either normal, swollen, or swollen with defective cristae (class I, II, or III; Figures 8D to 8F). In nfatc3+/+nfatc4+/+ E10.5 cardiomyocytes, only 16.5% of the mitochondria were abnormal class II (Figure 8D). Similar levels of normal mitochondria were observed in nfatc3+/+nfatc4+/+/NFATc4{Delta}317 control embryos (data not shown). Conversely, class II and III mitochondria constituted 40.4% of the mitochondria observed in nfatc3-/-nfatc4-/- E10.5 cardiomyocytes. Cardiac-specific expression of NFATc4{Delta}317 partially restored the mitochondrial phenotype in the nfatc3-/-nfatc4-/- background, increasing the percentage of normal mitochondria to >70% (Figure 8E). Stereological analysis was used to further characterize the mitochondrial defects in relation to cell volume and mitochondrial size. Although the total number of mitochondria observed was comparable between the genotypes, the percentage of the total cytoplasmic volume consisting of mitochondria was doubled in nfatc3-/-nfatc4-/- embryos compared with nfatc3+/+nfatc4+/+ embryos (see online Figure 2A, available at http://www.circresaha.org). The increase in mitochondrial volume corresponds to a doubling of the average mitochondrial size in nfatc3-/-nfatc4-/- ventricular cardiomyocytes (see online Figure 2B). Thus, restoration of NFAT activity in nfatc3-/-nfatc4-/- embryonic hearts resulted in the preservation of the mitochondrial activity, morphology, average mitochondrial size, and cytoplasmic volume used. These data, together with the proliferation and histological analysis, confirm that restoration of cardiac-specific NFAT activity rescues nfatc3-/-nfatc4-/- embryonic lethality at {approx}E11 and suggest that NFAT regulation of metabolism is an essential aspect of cardiac development.



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  		Figure 8. NFATc4{Delta}317 expression improves mitochondrial function and morphology in nfatc3-/-nfatc4-/- embryos. A through C, SDH enzyme activity, as detected by nitro blue tetrazolium precipitation, is restored to {approx}100% activity in nfatc3-/-nfatc4-/-/NFATc4{Delta}317 E10.5 hearts (B, n=2) compared with the {approx}30% reduction in nfatc3-/-nfatc4-/- whole hearts (C). D through F, Mitochondria were classified as follows: class I, normal (D); class II, malformed cristae (E); and class III, swollen, few cristae (F). Representative TEMs of nfatc3+/+nfatc4+/+ (D), nfatc3-/-nfatc4-/-/NFATc4{Delta}317 (E), and nfatc3-/-nfatc4-/- (F) mitochondria are shown. In panel F, 40% of the mitochondria in nfatc3-/-nfatc4-/- cardiomyocytes are class II, with {approx}15% being class III. In panel E, cardiac-specific restoration of NFAT signaling in the nfatc3-/-nfatc4-/- background maintains a substantial number of mitochondria as class I and reduces the number of class III deformed mitochondria.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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The requirement for NFAT activity in cardiac development was examined in mice deficient for NFATc3 and NFATc4. Nfatc3-/-nfatc4-/- embryos exhibited embryonic lethality by {approx}E11, with cardiac abnormalities indicative of heart failure. Cardiomyocyte differentiation was not affected by the loss of NFAT activity, as evidenced by sarcomere formation, cardiomyocyte beating, and ß-MyHC expression. However, impaired cardiac development was apparent in the thin ventricular myocardial compact zone and reduction in trabeculation. Increased cell death was not apparent in nfatc3-/-nfatc4-/- embryos, whereas proliferation was decreased by >50%. Additionally, the mitochondria in nfatc3-/-nfatc4-/- embryonic hearts were swollen, with abnormal cristae and reduced metabolic activity. A primary defect in mitochondrial oxidative capacity was observed in cultured cardiomyocytes isolated from nfatc3-/-nfatc4-/- embryos at E9.5, a day before any observed cardiovascular abnormalities. It is likely that the compromised mitochondria of nfatc3-/-nfatc4-/- hearts are incapable of meeting the increased circulatory requirements of the E10.5 embryos, leading to cardiovascular failure and death in {approx}0.5 days. Restoration of cardiac NFAT activity rescued embryonic viability, cardiomyocyte proliferation, mitochondrial morphology, and oxidative function, indicating that the cardiac defects in nfatc3-/-nfatc4-/- embryos were the primary cause of death. Ventricular NFATc4{Delta}317 expression was lost at E11.5, and the embryos died within {approx}0.5 day. Thus, continued cardiac NFAT expression is required for embryonic survival.

Activation of NFAT plays a key role in muscle development and adaptation to environmental stress.1,5,8 Overexpression of NFATc4 in adult mice is sufficient to induce cardiac hypertrophy,9 and NFATc3 deficiency significantly reduces pathophysiological and CNA-induced hypertrophy.20 NFATc3 and NFATc4 directly regulate the expression of metabolic proteins, including adenyl succinate synthase 1 and carnitine palmitoyltransferase 1, through interaction with GATA-4 and myocyte enhancer factor 2C in cardiomyocytes.10,11,17,18 In skeletal muscle, CNA signaling and NFAT activity promotes differentiation of slow-twitch fibers, which have a higher rate of oxidative metabolism and elevated levels of intracellular Ca2+ relative to fast-twitch fibers.10,11 NFATc3 and NFATc4 are able to induce fast-to-slow fiber transformation, whereas inhibition of CNA by cyclosporine promotes slow-to-fast fiber transformation. Supporting evidence for CNA/NFAT regulation of cardiac metabolism is provided by transgenic mice with cardiac expression of constitutively active CNA that have mitochondrial dysfunction and elevated superoxide production in cardiomyocytes.31 Together, these data suggest that NFAT has a regulatory role in cardiac and skeletal muscle metabolism and mitochondrial function. This conclusion is corroborated by the reduced respiratory activity and compromised mitochondrial ultrastructure observed in nfatc3-/-nfatc4-/- cardiomyocytes.

A critical component of cardiovascular development is the establishment of a metabolic system capable of sustaining continuous high levels of mechanical activity. The heart is the first functional organ in the developing embryo, with contractions starting soon after formation of the linear heart tube and continuing throughout life.32 Thus, metabolic pathways that supply the high level of required energy must be established relatively early during embryogenesis.14,33 Whereas early-stage embryos are primarily dependent on glycolysis, oxidative phosphorylation is the predominant source of cellular energy by midgestation, and loss of critical mitochondrial transcription factors or metabolic genes leads to midgestational embryonic lethality.14,16,34,35 The reduction in CoxC and SDH enzyme activity and the primary defects in mitochondrial oxidative capacity observed in nfatc3-/-nfatc4-/- cardiomyocytes are indicative of compromised energy metabolism in the absence of NFATc3 and NFATc4. Similarly, targeted disruption of the Pex5 gene, important for ß-oxidation, results in pleomorphic mitochondria, abnormal cristae, and megamitochondria with reduced metabolic activity and ATP production.23 Mitochondrial structural and functional defects are also described with ethanol toxicity, known to result in cardiomyopathy and reduced mitochondrial activity and energy production.25,36 The mitochondrial phenotypes of both Pex5-/-- and ethanol-treated mice are comparable to class III mitochondria of nfatc3-/-nfatc4-/- embryos, which also have reduced mitochondrial CoxC and SDH activity. The coordinate loss of cardiac tissue and metabolic capacity offers a rationale for the E10.5 embryonic lethality observed in nfatc3-/-nfatc4-/- embryos when circulation is essential for life.

Development of a functional cardiovascular system must anticipate the circulatory requirements of the embryo and is essential for embryonic survival by midgestation. During the early stages of embryogenesis, oxygen, nutrients, and metabolites freely diffuse between the maternal blood supply and the developing embryo. A critical time point in the diffusion/circulation transition occurs at midgestation, {approx}E10.5 in mice, during maturation of the ventricles.32 Several lines of mice die at this time, including nfatc3/nfatc4, nkx2.5, Ncx-1, and N-cadherin–deficient mice.6,37–39 Development of the cardiovascular system is dependent on the coordinated maturation of the heart and blood vessels; therefore, midgestation embryonic lethality could be due to defects in either system. The interdependence of heart and vascular development is seen in the N-cadherin null mice,39 which have cardiovascular defects and die at {approx}E10.5. The primary cause of death in these embryos is lack of N-cadherin in the heart, because cardiac-specific restoration of N-cadherin restores cardiac development, vascular integrity, and embryonic viability. Additionally, the impaired development of yolk sac vessels in Ncx-1 mutant mice is secondary to the loss of heartbeats and insufficient hemodynamics.38 These data demonstrate the requirement of heart development for proper vascular maturation. Similarly, nfatc3-/-nfatc4-/- embryos have cardiovascular defects. Graef et al6 recently reported vascular defects in E10.5 nfatc3-/-nfatc4-/- embryos, with disorganized vessel growth and loss of major blood vessel integrity. Using a different nfatc4-/- mouse line,20 we report in the present study that E10.5 nfatc3-/-nfatc4-/- embryos have thin dilated ventricles, reduced trabeculation, and dysmorphic mitochondria and that they die at {approx}E11. Given the developmental interdependence of the cardiac and vascular systems,37–39 these two sets of data are not mutually exclusive. However, cardiac-specific restoration of NFAT activity restores ventricular maturation and proliferation and prolongs embryonic life, indicating that the cardiac defects in nfatc3-/- nfatc4-/- embryos are the primary cause of lethality at {approx}E11.

Combinatorial disruption of nfatc3 and nfatc4 results in defective cardiac development, dysfunctional ventricular mitochondria, and lethality at {approx}E11. Expression of the vast majority of genes tested involved in metabolism and mitochondrial biogenesis was unchanged in nfatc3-/-nfatc4-/- embryos, with the exceptions of heart–fatty acid binding protein and pyruvate carboxylase. Interestingly, multiple NFAT consensus binding sequences are present in cardiac regulatory sequences of the human HFABP gene.28 Further studies are necessary to identify the direct transcriptional targets by which NFATs regulate cardiac mitochondrial morphology and function. However, the analysis of nfatc3/nfatc4 mutant embryos demonstrates the requirement for these genes in maintaining embryonic viability. The midgestation metabolic transition represents a critical window of development in the formation of an effective cardiovascular system. This process can be disrupted by a variety of genetic or teratological agents, including mutation of nkx2.5,37 N-cadherin,39 diabetic hyperglycemia,24 or ethanol toxicity.25,36 The identification of critical regulators of embryonic cardiac metabolic transitions, such as NFATs, may provide future therapeutic strategies for treating congenital heart defects associated with mitochondrial dysfunction as a result of ethanol consumption or inborn errors in metabolism.


*    Acknowledgments
 
This study was supported by NIH grant PO1-HL-069779 (Drs Yutzey and Molkentin) and an Established Investigator Award from the American Heart Association (Dr Yutzey). Support for Dr Bushdid was provided by NIH grants T32-ES-07051 and F32-HL-71370. We thank Jeff Robbins, Christina Alfieri, Alex Lange, Christine Liberatore, T.J. Plageman, and the Division of Molecular Cardiovascular Biology for critical discussions and technical advice.


*    Footnotes
 
Original received December 27, 2002; revision received May 1, 2003; accepted May 5, 2003.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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