Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability
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《循环研究杂志》
the Department of Pediatrics (T.O., M.M., B.J.A., J.D.M), University of Cincinnati, Division of Molecular Cardiovascular Biology, Children’s Hospital Medical Center, Ohio
Department of Cell Biology, Neurobiology
Anatomy (A.J.W., S.A.D.), Medical College of Wisconsin, Milwaukee
The Institute of Biosciences and Technology (R.J.S.), Texas A&M University System Health Science Center, Houston.
Abstract
The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxPeCtargeted mice (Gata4fl/fl) were crossed with mice containing a -myosin heavy chain (-MHC) or -MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.
Key Words: heart transcription hypertrophy mouse genetics apoptosis
Introduction
GATA4 is a zinc finger-containing transcription factor that plays an essential role in promoting cardiac development and differentiation of the myocardium, as well as in regulating survival and hypertrophic growth of the adult heart.1,2 GATA4 is highly expressed in both embryonic and adult cardiomyocytes where it is thought to function as a key transcriptional regulator of numerous cardiac genes including atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), -myosin heavy chain (-MHC), -MHC, and many others.1,2 A direct transcriptional regulatory role for GATA4 is further supported by the observation that antisense GATA4 mRNA expression inhibited the basal expression of certain cardiac-expressed genes in cardiomyocyte cultures.3 In addition to its hypothesized role in maintaining differentiated gene expression in the heart, GATA4 also mediates inducible gene expression in response to hypertrophic stimuli, including pressure overload, isoproterenol, phenylephrine, and endothelin-1.4eC7 That GATA4 is sufficient to induce cardiac hypertrophy was demonstrated by overexpression of GATA4 in cultured cardiomyocytes and transgenic mice.7 Moreover, expression of a dominant negative GATA4 or antisense GATA4 mRNA blocked features of cardiomyocyte hypertrophy induced by phenylephrine and endothelin-1 in culture.7,8
A number of stimuli that induce cardiac hypertrophy and/or heart failure were shown to enhance GATA4 transcriptional activity through phosphorylation. For example, pressure overload, isoproterenol, phenylephrine, endothelin-1, angiotensin II, and phorbol esters induced phosphorylation of GATA4, resulting in enhanced DNA binding and/or transactivating activity.4,6,9eC13 We have previously observed that agonist stimulation results in phosphorylation of GATA4 at serine 105 through the direct action of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK).8,10 Phosphorylation of serine 105 in GATA4 enhanced DNA binding activity and transcriptional potency, whereas mutation of serine 105 to alanine attenuated activity.10 Because both ERK1/2 and p38 MAPK receive input from diverse upstream signaling pathways, it suggested that serine 105 in GATA4 is a key convergence point in regulating the cardiac hypertrophic response. Finally, GATA4 is also subjected to negative regulation by glycogen synthase kinase 3, which was shown to reduce both basal and isoproterenol-induced nuclear expression of GATA4 and to suppress GATA4 transcriptional activity.11
More recently, the requirement of GATA4 in regulating cardiac development and differentiation of myocytes has been investigated in genetically modified mice, although these studies did not examine the importance of GATA4 in regulating hypertrophy and/or heart failure in the adult. Using a tetraploid rescue strategy, Gata4eC/eC embryos showed disrupted looping morphogenesis and a hypoplastic ventricular myocardium.14 Similarly, conditional disruption of Gata4-loxPeCtargeted alleles in the heart using a Cre-loxP approach in conjunction with a Nkx2.5-Cre"knock-in" allele resulted in embryonic lethality associated with disrupted cardiac development and hypoplastic ventricles.15,16 Moreover, human mutations in Gata4 are associated with congenital abnormalities of the heart, collectively suggesting that GATA4 is a critical regulator of heart development.17 Here we investigated the importance of GATA4 in regulating the maintenance and compensatory responsiveness of the adult heart using a Gata4-loxP-targeted allele (Gata4fl/fl) in conjunction with 3 different cardiac-expressing Cre alleles/transgenes. Loss of Gata4 from the adult heart severely compromised basal gene expression, survival of cardiac myocytes, and the ability of the myocardium to hypertrophy and compensate to pressure overload or hypertrophy following exercise stimulation.
Materials and Methods
Genetically Modified Mice
The generation and characterization of Gata4fl/fl-targeted embryonic stem cells and mice, in which exons 3, 4, and 5 were flanked with loxP sites, was described previously.14 Gata4fl/fl mice and their controls were maintained in the SV129/CD1 genetic background (Gata4tm1Sad, http://www.informatics.jax.org/javawi2/servlet/WIFetchpage=alleleDetail&key=27673). The -myosin heavy chain (-MHC) promoter-driven Cre transgenic line was described previously.18 The Nkx2.5-Cre knock-in allele was also described previously.19 Transgenic mice were also generated in which a nuclear-localizing Cre cDNA was placed under the control of the mouse -MHC promoter.20 All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center.
Pressure Overload by Transverse Aortic Constriction
Eight week-old mice were subjected to transverse aortic constriction (TAC) under isoflurane anesthesia as previously described.21 Pressure gradients (PG) (mm Hg) were calculated from the peak blood velocity (Vmax) (m/s) (PG=4 Vmax2) measured by Doppler across the aortic constriction. Body and ventricle weights were measured to calculate ventricle weight to body weight ratios (VW/BW) (mg/g) 2 and 4 weeks after TAC.
Histology, Immunohistochemistry, Echocardiography, Western Blotting, Hydroxyproline Content Determination, Swimming, Affymetrix Gene Expression Profiling, Bioinformatics, and RT-PCR Analysis
For additional details regarding the methods used, see the expanded Materials and Methods section and supplemental Table I, both available in the online data supplement at http://circres.ahajournals.org.
Statistics
Data are expressed as mean±SEM. Statistical significance between 2 groups was determined with a paired Student’s t test. Probability values of <0.05 were considered statistically significant.
Results
Tissue-Specific Deletion of Gata4 in the Heart
Standard Gata4 gene-targeted mice perish during early embryonic development precluding an analysis of GATA4’s function in the adult heart.22,23 To address this limitation here, we used a Cre-loxP-dependent conditional gene targeting approach to permit specific inactivation of Gata4 in the heart at later developmental stages.14 Mice homozygous for the Gata4-loxPeCtargeted allele (fl/fl) were crossed with transgenic mice expressing Cre from the -MHC promoter. Gata4fl/flMHC-Cre mice were generated at predicted Mendelian ratios and were viable as young adults up to 16 weeks of age, although by 40 weeks of age approximately 25% had expired (data not shown). Inspection of embryonic hearts by immunohistochemistry showed no appreciable reduction in GATA4 protein levels in the developing myocardium at embryonic day (E) 12.5 in Gata4fl/flMHC-Cre embryos compared with Gata4fl/fl embryos (Figure 1A, 1B, 1E, 1F, and 1I). However, a greater than 70% reduction in GATA4 positive nuclei was observed by E17.5 (Figure 1C, 1D, and 1G through 1I). Gata4fl/fl mice showed no reduction in GATA4 protein compared with wild-type controls, indicating that the placement of loxP sites within the Gata4 locus did not alter expression of the GATA4, in contrast to another description of Gata4-loxPeCtargeted mice that were hypomorphic for GATA4 expression based on different loxP site placements in the Gata4 locus.15
By 8 weeks of age, approximately 95% of all myocardial nuclei were devoid of GATA4 protein in Gata4fl/flMHC-Cre mice compared with control mice (Figure 2A through 2D and 2G). Deletion of GATA4 protein expression in these mice was homogenous throughout the ventricles and atrial myocardial cells but excluded the endocardium (data not shown). Lastly, an additional Cre-expressing transgenic line that used the -MHC promoter was also generated (Gata4fl/flMHC-Cre), which also produced viable adults and demonstrated a 70% deletion of GATA4 protein in myocardial cells at 8 weeks of age (Figure 2E, 2F, and 2G). Western blotting from total heart nuclear extracts at 8 weeks of age, although not specific for cardiac myocytes, showed a similar reduction in GATA4 protein by each Cre transgene compared with immunohistochemistry (Figure 2H). Although both - and -MHC promoters are expressed at different times and intensities in the murine heart during development, the fact that both transgenes produce viable adults in the Gata4fl/fl background suggests that temporal expression differences are less important and that the primary difference between the 2 Cre transgenes likely relates to the cumulative extent of Gata4 deletion.
Deletion of Gata4 From the Adult Heart Compromises Function
Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice were each followed for functional alterations in cardiac performance by echocardiography up to 24 weeks of age. At 12 weeks of age, Gata4fl/flMHC-Cre mice showed a significant reduction in fractional shortening that progressively worsened up to 24 weeks of age (Figure 3A and Table 1). None of the control groups showed any significant reduction in fractional shortening, indicating that the Gata4-loxP allele itself and the Cre transgenes were innocuous (Figure 3A and Table 1). Gata4fl/flMHC-Cre mice also showed significant increases in left ventricular end-systolic and -diastolic dimensions, indicating ventricular dilation (Table 1). Consistent with the slightly less robust deletion of Gata4 mediated by the -MHC-Cre transgene, Gata4fl/flMHC-Cre mice did not show a significant reduction in fractional shortening until 24 weeks of age (Figure 3A, Table 1). Taken together, these data indicate that loss of GATA4 expression from the heart compromises cardiac function and induces dilation.
To more carefully evaluate the importance of GATA4 protein in the maintenance of proper heart structure and function, pressure overload (TAC) stimulation was performed in 8-week-old mice and followed for 1, 2, 3, and 4 weeks. Remarkably, TAC rapidly and progressively induced decompensation and dilation in Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice compared with no effect in any of the control groups (Figure 3B and Table 2). Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, but not controls, showed severe pulmonary edema following TAC (lung to body weight ratio: 20.45±0.71 mg/g in Gata4fl/flMHC-Cre; 18.51±0.70 mg/g in Gata4fl/flMHC-Cre; and 10.35±0.91 mg/g in controls [P<0.05]). Consistent with the total extent of GATA4 protein deletion in the myocardium, Gata4fl/flMHC-Cre showed a more severe profile of decompensation and dilation compared with Gata4fl/flMHC-Cre mice (Figure 3B and Table 2). Also of interest, all groups showed a similar surgical lethality profile (10%), although 3 of 23 Gata4fl/flMHC-Cre mice subjected to TAC died within the postsurgical period, whereas none of the other groups showed any lethality in this period (supplemental Table II). Collectively, these results indicate that GATA4 is required for compensation of heart performance following pressure overload stimulation.
Deletion of Gata4 Attenuates the Cardiac Hypertrophic Response
Although GATA4 has been implicated as a mediator of the cardiac hypertrophic response, a loss-of-function approach has yet to be evaluated in vivo. Here we examined the extent of cardiac hypertrophy by assessment of VW/BW and by measurement of cell areas in heart histological sections from TAC mice after 4 weeks of stimulation. Both Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice showed a significant 40% to 50% reduction in the hypertrophic response compared with each of the control groups subjected to TAC for 2 or 4 weeks (Figure 4A and data not shown). However, Gata4fl/flMHC-Cre TAC mice showed no enhancement in histopathology over wild types (data not shown), and fibrosis was not significantly greater (Figure 4B). At the cellular level, Gata4fl/flMHC-Cre mice showed a significant reduction in cardiomyocyte area following TAC for 4 weeks (Figure 4C). Interestingly, cardiac histological sections were also costained for GATA4 from Gata4fl/flMHC-Cre mice, and the 30% of residual cells with GATA4 protein expression were significantly larger after TAC compared with neighboring cells within the same heart that were GATA4-deficient (Figure 4C). These results suggest that GATA4 functions in a cell autonomous manner to regulate hypertrophy. Echocardiography-based assessment of ventricular wall thicknesses also showed attenuated growth in Gata4fl/flMHC-Cre mice compared with controls (Table 2). Mice were also subjected to Doppler echocardiography for analysis of transaortic pressure gradients developed across the constriction, which showed no differences between groups (data not shown). Lastly, Gata4fl/flMHC-Cre mice were also subjected to swimming to evaluate exercise-induced cardiac hypertrophy, which showed significantly less growth compared with wild-type and -MHC-Cre mice (Figure 4D). Collectively, these results indicate that GATA4 is a necessary mediator of the cardiac hypertrophic response in vivo.
Loss of Gata4 Increases Cell Death in the Heart
The observation that Gata4fl/flMHC-Cre mice showed baseline ventricular dilation and a progressive reduction in cardiac functional performance with age suggested that cell death might be involved. Indeed, Gata4+/eC mice were previously shown to have enhanced cell death in the heart following doxorubicin treatment.24 To determine whether enhanced cell death might partially underlie the cardiac phenotype described above, TUNEL was performed in adult and embryonic hearts from Gata4fl/flMHC-Cre and Gata4fl/flNkx2.5-Cre mice, respectively. As speculated, baseline TUNEL was significantly increased in Gata4fl/flMHC-Cre mice compared with wild-type and -MHC-Cre mice alone at 12 and 20 weeks of age (Figure 5A). Moreover, TAC stimulation for 4 weeks induced a greater increase in TUNEL in Gata4fl/flMHC-Cre mice compared with the control groups (P<0.05) (Figure 5A). These results suggest that loss of cardiac myocytes at baseline and following pressure overload stimulation contributes to the decompensation observed in Gata4fl/flMHC-Cre mice.
To more thoroughly evaluate the hypothesis that loss of GATA4 enhances cardiac TUNEL within another context, embryos from Gata4-loxPeCtargeted mice crossed with the Nkx2.5-Cre knock-in allele were examined at E12.5.19 The Nkx2.5-Cre allele produces much earlier expression of Cre in the heart, resulting in a greater than 95% loss of GATA4 protein from the myocardium by E12.5 of development (Figure 5B). Targeting of Gata4 in this manner resulted in embryonic lethality between E12.5 and E15.5, with a uniform reduction in ventricular wall thicknesses (data not shown). However, a more extensive description of the embryonic phenotype is not presented here because it has been given by 2 other groups.14eC16 Consistent with the observations made in the adult heart, Gata4fl/flNkx2.5-Cre embryos showed significantly higher rates of TUNEL in the heart compared with Gata4fl/fl or Gata4fl/flMHC-Cre embryos, the latter of which had not yet lost significant GATA4 protein expression (Figures 5C and 1F). Thus, loss of GATA4 from the adult or embryonic heart likely enhances myocyte apoptosis levels.
Loss of Gata4 From the Heart Dramatically Alters Basal Gene Expression
Although cellular apoptosis may partially explain the mechanistic underpinnings of decompensation and lethality in Gata4 heart-targeted mice, the exact molecular mechanism was uncertain. Because GATA4 is a transcription factor, the obvious hypothesis is that a subset of cardiac-expressed genes involved in cellular death and/or decompensation would be altered. This hypothesis was evaluated in an unbiased manner by array screening with the Affymetrix MOE 430 to 2 chip set using RNA from the hearts of 8-week-old Gata4fl/flMHC-Cre mice and 2 different control groups (Figure 6A). Eight weeks was selected for generation of RNA because this time largely precedes any pathological manifestations, so that secondary alterations in gene expression were less likely, and, at this time, there was no basal activation of select signaling kinases involved in cardiac stress stimulation (supplemental Figure). Compared with both control groups, hearts from Gata4fl/flMHC-Cre mice showed a significant change in 362 genes compared with cardiac RNA from wild-type mice, which is approximately 1.0% of all surveyed genes (Figure 6A). Most notably, GATA4 was previously shown to directly regulate expression of the ANF, -MHC, and -MHC genes, each of which is significantly altered in the hearts of Gata4fl/flMHC-Cre mice (Figure 6B). Interestingly, 3 genes with a known role in regulating apoptosis were also significantly altered in the absence of Gata4 (Figure 6B and 6C). For example, protein kinase C (PKC) expression was reduced in Gata4-deleted hearts, which based on previous literature, should enhance cell death.25 Similarly, Gata4-deleted hearts showed an upregulation in expression of the proapoptotic factor caspase-12 (csp12) and Bcl6. Gata4-deleted hearts also had significant alterations in expression of other signaling and transcriptional regulatory factors such as Sox7, Iroquois-related homeobox gene 3 (Irx3), fibroblast growth factor 1 (FGF1), and prostaglandin F receptor (PGFR) (Figure 6B and 6C). In conclusion, targeted deletion of Gata4 from the myocardium leads to profound alterations in cardiac gene expression, a subset of which might partially underlie the observed increase in myocyte TUNEL or ventricular decompensation and loss of hypertrophic potential.
Discussion
GATA4 has been ascribed to a number of critical functions in the heart, spanning from the specification and differentiation of cardiac myocytes early in development to the regulation of the cardiac hypertrophic response in the adult. GATA4 mediates these processes through the direct transcriptional control of key cardiac structural and regulatory genes.1 For example, GATA4 has been shown to directly bind the promoters of the ANF, BNP, -MHC, and -MHC genes, thereby controlling their expression in the heart.1 Indeed, 3 of these 4 genes showed altered expression in the hearts of Gata4fl/flMHC-Cre mice, validating the previously proposed proximal regulatory role of GATA4. Cardiac-specific deletion of Gata4 also resulted in the dysregulation of approximately 1.0% of all cardiac-expressed genes, further supporting the overall importance of GATA4 as a homeostatic regulator of gene expression in the heart.
GATA4 is also thought to function as a critical regulator of cardiac hypertrophy, although most studies conducted to date relied on cultured neonatal cardiac myocytes as a model system. For example, overexpression of GATA4 in culture by adenoviral gene transfer induced cardiomyocyte hypertrophy, indicating the sufficiency of GATA4 in this process.7 More significantly, expression of dominant negative GATA4 (engrailed fusion) or antisense GATA4 mRNA each blocked GATA4-directed transcriptional responses and features of cardiomyocyte hypertrophy induced by phenylephrine and endothelin-1 in culture.7,8 In vivo, mild overexpression of GATA4 in the mouse heart by transgenesis induced a progressive hypertrophic response.7 However, direct assessment of the necessity of GATA4 in mediating cardiac hypertrophy in vivo had not been examined. Here we demonstrated for the first time that reduction in GATA4 expression by 70% or 95%, specifically within cardiac myocytes, attenuated the cardiac hypertrophic response following 2 or 4 weeks of pressure overload or following exercise stimulation in Gata4fl/flMHC-Cre mice. Interestingly, the 95% reduction in total GATA4 positive cardiac myocytes induced by the -MHC-Cre transgene showed a more consistent reduction in cardiac hypertrophy and earlier functional decompensation (Figure 3A and 3B and Table 2) compared with a 70% reduction in GATA4 positive cardiac myocytes induced by the -MHC-Cre transgene, suggesting a dosage-dependent effect. It is also interesting to note that cardiac-specific deletion of Gata4 only reduced the cardiac hypertrophic response by approximately 40% to 50%, suggesting either that other regulators are sufficient to mediate some degree of cardiac hypertrophy in the absence of Gata4 or that Gata6 can partially compensate for the loss of Gata4 in the adult heart (Gata5 is not expressed in the adult heart). However, loss of GATA4 did not affect postnatal growth of the heart, also referred to as developmental hypertrophy, suggesting that different transcriptional regulators are involved in this process, or that GATA6 can compensate for this type of growth. With respect to functional decompensation after pressure overload, the phenotype of Gata4-deleted mice is reminiscent of melusin gene targeting, which is a muscle-specific 1-integrin interacting protein. Loss of melusin compromised the hypertrophic response and promoted functional decompensation, in contrast to the phenotype of Gq-inhibited transgenic mice or dopamine -hydroxylase gene-targeted mice, which had inhibited cardiac hypertrophy after pressure overload without functional decompensation.26,27
Traditional germline disruption of Gata4 in the entire mouse resulted in early embryonic lethality between E7.0 and E9.5 because of defects in endoderm and ventral morphogenesis.22,23 However, these embryos still generated cardiac tissue that expressed heart-specific structural genes, suggesting that GATA4 was not required for specification and efficient differentiation, although GATA5 and GATA6, which are each expressed in the early developing cardiac splanchnic mesoderm, likely compensated.22,23 More recently, 2 additional approaches were used to gain insight into the role of GATA4 in later aspects of cardiac development. Tetraploid embryo complementation was employed using Gata4eC/eC embryonic stem cells, which generated embryos that progressed further in development and showed hypoplastic ventricles and a loss of the proepicardium, resulting in lethality.14 Gata4 was also deleted specifically in the heart by Pu and colleagues using a Cre-loxP-based approach and the same Nkx2.5-Cre knock-in allele that we used.15 However, the loxP insertion sites selected by Pu et al produced a hypomorphic Gata4 allele that reduced basal expression by approximately 50%.15 These Gata4fl/fl mice showed early embryonic lethality when crossed into with the Nkx2.5-Cre allele, and later embryonic lethality when crossed with an -MHC-Cre transgene.15,16 In contrast, our Gata4-loxP targeted allele did not disrupt basal expression so that both Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice were viable and amenable to analysis as adults. However, consistent with Pu et al, Gata4fl/flNkx2.5-Cre mice showed embryonic lethality, but at a slightly later time point with our Gata4-loxPeCtargeting strategy (E12.5-E15.5). The reason underlying lethality in Gata4fl/flNkx2.5-Cre embryos, but not Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, is likely attributable to the much earlier profile of Nkx2.5-Cre expression compared with the -MHC and -MHC transgenes.
Mechanistically, adult hearts from Gata4fl/flMHC-Cre mice showed greater TUNEL at baseline, as well as significantly enhanced levels of TUNEL following pressure overload stimulation. Moreover, hearts from Gata4fl/flNkx2.5-Cre embryos at E12.5 also showed enhanced TUNEL compared with Gata4fl/flMHC-Cre embryos, which do not have appreciable GATA4 reductions until later in development. Thus, loss of Gata4 from the embryonic or adult heart predisposes to myocyte loss by apoptosis, likely contributing to the observed ventricular thinning in adults and hypoplastic ventricles in embryos. However, Pu and colleagues also reported a reduction in cellular proliferation in the absence of Gata4 in the developing heart, which could also play an important role.16 Our observations of increased TUNEL in the absence of Gata4 is consistent with the results of Aries et al, in which Gata4+/eC mice (heterozygotes) showed greater functional decompensation following doxorubicin treatment and greater TUNEL in cultured cardiomyocytes infected with an antisense mRNA expressing GATA4 adenovirus following doxorubicin treatment.24 By comparison, doxorubicin-mediated cell death in cultured cardiomyocytes or HL-1 cells was reversed by GATA4 or GATA6 overexpression.28 Moreover, depletion of GATA4 from chicken embryos enhanced cellular apoptosis in the heart-forming region and underlying endoderm.29 Collectively, these results suggest that GATA4 regulates, in part, cellular viability at baseline and in response to stress stimulation.
However, it is uncertain whether cellular attrition through apoptosis directly underlies the progressive decompensation that was observed in Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, as we were unable to directly quantify total cell number in adult hearts of the various genotypes examined here. It is also formally possible that many of the profound alterations in gene expression that occurs in the absence of Gata4 could underlie the propensity toward decompensation without a loss in myocyte number. Indeed, the dramatic shift from -MHC to -MHC expression, as observed in the hearts of Gata4fl/flMHC-Cre mice, would be predicted to reduce the contractile performance of the myocardium, hence leading possibly to greater decompensation after stress stimulation. However, it is likely that a progressive loss of cardiac myocytes, together with the observed alterations in cardiac structural and regulatory gene expression, leads to the observed phenotype in cardiac-specific Gata4-deleted mice.
Acknowledgments
This work was supported by grants from the NIH, an American Heart Association Established Investigator Grant (to J.D.M), and an American Heart Association Postdoctoral Fellowship grant (0425393B to T.O.).
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Ghatpande S, Ghatpande A, Zile M, Evans T. Anterior endoderm is sufficient to rescue foregut apoptosis and heart tube morphogenesis in an embryo lacking retinoic acid. Dev Biol. 2000; 219: 59eC70.(Toru Oka, Marjorie Maille)
Department of Cell Biology, Neurobiology
Anatomy (A.J.W., S.A.D.), Medical College of Wisconsin, Milwaukee
The Institute of Biosciences and Technology (R.J.S.), Texas A&M University System Health Science Center, Houston.
Abstract
The transcription factor GATA4 is a critical regulator of cardiac gene expression where it controls embryonic development, cardiomyocyte differentiation, and stress responsiveness of the adult heart. Traditional deletion of Gata4 caused embryonic lethality associated with endoderm defects and cardiac malformations, precluding an analysis of the role of GATA4 in the adult myocardium. To address the function of GATA4 in the adult heart, Gata4-loxPeCtargeted mice (Gata4fl/fl) were crossed with mice containing a -myosin heavy chain (-MHC) or -MHC promoter-driven Cre transgene, which produced viable mice that survived into adulthood despite a 95% and 70% loss of GATA4 protein, respectively. However, cardiac-specific deletion of Gata4 resulted in a progressive and dosage-dependent deterioration in cardiac function and dilation in adulthood. Moreover, pressure overload stimulation induced rapid decompensation and heart failure in cardiac-specific Gata4-deleted mice. More provocatively, Gata4-deleted mice were compromised in their ability to hypertrophy following pressure overload or exercise stimulation. Mechanistically, cardiac-specific deletion of Gata4 increased cardiomyocyte TUNEL at baseline in embryos and adults as they aged, as well as dramatically increased TUNEL following pressure overload stimulation. Examination of gene expression profiles in the heart revealed a number of profound alterations in known GATA4-regulated structural genes as well as genes with apoptotic implications. Thus, GATA4 is a necessary regulator of cardiac gene expression, hypertrophy, stress-compensation, and myocyte viability.
Key Words: heart transcription hypertrophy mouse genetics apoptosis
Introduction
GATA4 is a zinc finger-containing transcription factor that plays an essential role in promoting cardiac development and differentiation of the myocardium, as well as in regulating survival and hypertrophic growth of the adult heart.1,2 GATA4 is highly expressed in both embryonic and adult cardiomyocytes where it is thought to function as a key transcriptional regulator of numerous cardiac genes including atrial natriuretic factor (ANF), b-type natriuretic peptide (BNP), -myosin heavy chain (-MHC), -MHC, and many others.1,2 A direct transcriptional regulatory role for GATA4 is further supported by the observation that antisense GATA4 mRNA expression inhibited the basal expression of certain cardiac-expressed genes in cardiomyocyte cultures.3 In addition to its hypothesized role in maintaining differentiated gene expression in the heart, GATA4 also mediates inducible gene expression in response to hypertrophic stimuli, including pressure overload, isoproterenol, phenylephrine, and endothelin-1.4eC7 That GATA4 is sufficient to induce cardiac hypertrophy was demonstrated by overexpression of GATA4 in cultured cardiomyocytes and transgenic mice.7 Moreover, expression of a dominant negative GATA4 or antisense GATA4 mRNA blocked features of cardiomyocyte hypertrophy induced by phenylephrine and endothelin-1 in culture.7,8
A number of stimuli that induce cardiac hypertrophy and/or heart failure were shown to enhance GATA4 transcriptional activity through phosphorylation. For example, pressure overload, isoproterenol, phenylephrine, endothelin-1, angiotensin II, and phorbol esters induced phosphorylation of GATA4, resulting in enhanced DNA binding and/or transactivating activity.4,6,9eC13 We have previously observed that agonist stimulation results in phosphorylation of GATA4 at serine 105 through the direct action of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK).8,10 Phosphorylation of serine 105 in GATA4 enhanced DNA binding activity and transcriptional potency, whereas mutation of serine 105 to alanine attenuated activity.10 Because both ERK1/2 and p38 MAPK receive input from diverse upstream signaling pathways, it suggested that serine 105 in GATA4 is a key convergence point in regulating the cardiac hypertrophic response. Finally, GATA4 is also subjected to negative regulation by glycogen synthase kinase 3, which was shown to reduce both basal and isoproterenol-induced nuclear expression of GATA4 and to suppress GATA4 transcriptional activity.11
More recently, the requirement of GATA4 in regulating cardiac development and differentiation of myocytes has been investigated in genetically modified mice, although these studies did not examine the importance of GATA4 in regulating hypertrophy and/or heart failure in the adult. Using a tetraploid rescue strategy, Gata4eC/eC embryos showed disrupted looping morphogenesis and a hypoplastic ventricular myocardium.14 Similarly, conditional disruption of Gata4-loxPeCtargeted alleles in the heart using a Cre-loxP approach in conjunction with a Nkx2.5-Cre"knock-in" allele resulted in embryonic lethality associated with disrupted cardiac development and hypoplastic ventricles.15,16 Moreover, human mutations in Gata4 are associated with congenital abnormalities of the heart, collectively suggesting that GATA4 is a critical regulator of heart development.17 Here we investigated the importance of GATA4 in regulating the maintenance and compensatory responsiveness of the adult heart using a Gata4-loxP-targeted allele (Gata4fl/fl) in conjunction with 3 different cardiac-expressing Cre alleles/transgenes. Loss of Gata4 from the adult heart severely compromised basal gene expression, survival of cardiac myocytes, and the ability of the myocardium to hypertrophy and compensate to pressure overload or hypertrophy following exercise stimulation.
Materials and Methods
Genetically Modified Mice
The generation and characterization of Gata4fl/fl-targeted embryonic stem cells and mice, in which exons 3, 4, and 5 were flanked with loxP sites, was described previously.14 Gata4fl/fl mice and their controls were maintained in the SV129/CD1 genetic background (Gata4tm1Sad, http://www.informatics.jax.org/javawi2/servlet/WIFetchpage=alleleDetail&key=27673). The -myosin heavy chain (-MHC) promoter-driven Cre transgenic line was described previously.18 The Nkx2.5-Cre knock-in allele was also described previously.19 Transgenic mice were also generated in which a nuclear-localizing Cre cDNA was placed under the control of the mouse -MHC promoter.20 All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center.
Pressure Overload by Transverse Aortic Constriction
Eight week-old mice were subjected to transverse aortic constriction (TAC) under isoflurane anesthesia as previously described.21 Pressure gradients (PG) (mm Hg) were calculated from the peak blood velocity (Vmax) (m/s) (PG=4 Vmax2) measured by Doppler across the aortic constriction. Body and ventricle weights were measured to calculate ventricle weight to body weight ratios (VW/BW) (mg/g) 2 and 4 weeks after TAC.
Histology, Immunohistochemistry, Echocardiography, Western Blotting, Hydroxyproline Content Determination, Swimming, Affymetrix Gene Expression Profiling, Bioinformatics, and RT-PCR Analysis
For additional details regarding the methods used, see the expanded Materials and Methods section and supplemental Table I, both available in the online data supplement at http://circres.ahajournals.org.
Statistics
Data are expressed as mean±SEM. Statistical significance between 2 groups was determined with a paired Student’s t test. Probability values of <0.05 were considered statistically significant.
Results
Tissue-Specific Deletion of Gata4 in the Heart
Standard Gata4 gene-targeted mice perish during early embryonic development precluding an analysis of GATA4’s function in the adult heart.22,23 To address this limitation here, we used a Cre-loxP-dependent conditional gene targeting approach to permit specific inactivation of Gata4 in the heart at later developmental stages.14 Mice homozygous for the Gata4-loxPeCtargeted allele (fl/fl) were crossed with transgenic mice expressing Cre from the -MHC promoter. Gata4fl/flMHC-Cre mice were generated at predicted Mendelian ratios and were viable as young adults up to 16 weeks of age, although by 40 weeks of age approximately 25% had expired (data not shown). Inspection of embryonic hearts by immunohistochemistry showed no appreciable reduction in GATA4 protein levels in the developing myocardium at embryonic day (E) 12.5 in Gata4fl/flMHC-Cre embryos compared with Gata4fl/fl embryos (Figure 1A, 1B, 1E, 1F, and 1I). However, a greater than 70% reduction in GATA4 positive nuclei was observed by E17.5 (Figure 1C, 1D, and 1G through 1I). Gata4fl/fl mice showed no reduction in GATA4 protein compared with wild-type controls, indicating that the placement of loxP sites within the Gata4 locus did not alter expression of the GATA4, in contrast to another description of Gata4-loxPeCtargeted mice that were hypomorphic for GATA4 expression based on different loxP site placements in the Gata4 locus.15
By 8 weeks of age, approximately 95% of all myocardial nuclei were devoid of GATA4 protein in Gata4fl/flMHC-Cre mice compared with control mice (Figure 2A through 2D and 2G). Deletion of GATA4 protein expression in these mice was homogenous throughout the ventricles and atrial myocardial cells but excluded the endocardium (data not shown). Lastly, an additional Cre-expressing transgenic line that used the -MHC promoter was also generated (Gata4fl/flMHC-Cre), which also produced viable adults and demonstrated a 70% deletion of GATA4 protein in myocardial cells at 8 weeks of age (Figure 2E, 2F, and 2G). Western blotting from total heart nuclear extracts at 8 weeks of age, although not specific for cardiac myocytes, showed a similar reduction in GATA4 protein by each Cre transgene compared with immunohistochemistry (Figure 2H). Although both - and -MHC promoters are expressed at different times and intensities in the murine heart during development, the fact that both transgenes produce viable adults in the Gata4fl/fl background suggests that temporal expression differences are less important and that the primary difference between the 2 Cre transgenes likely relates to the cumulative extent of Gata4 deletion.
Deletion of Gata4 From the Adult Heart Compromises Function
Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice were each followed for functional alterations in cardiac performance by echocardiography up to 24 weeks of age. At 12 weeks of age, Gata4fl/flMHC-Cre mice showed a significant reduction in fractional shortening that progressively worsened up to 24 weeks of age (Figure 3A and Table 1). None of the control groups showed any significant reduction in fractional shortening, indicating that the Gata4-loxP allele itself and the Cre transgenes were innocuous (Figure 3A and Table 1). Gata4fl/flMHC-Cre mice also showed significant increases in left ventricular end-systolic and -diastolic dimensions, indicating ventricular dilation (Table 1). Consistent with the slightly less robust deletion of Gata4 mediated by the -MHC-Cre transgene, Gata4fl/flMHC-Cre mice did not show a significant reduction in fractional shortening until 24 weeks of age (Figure 3A, Table 1). Taken together, these data indicate that loss of GATA4 expression from the heart compromises cardiac function and induces dilation.
To more carefully evaluate the importance of GATA4 protein in the maintenance of proper heart structure and function, pressure overload (TAC) stimulation was performed in 8-week-old mice and followed for 1, 2, 3, and 4 weeks. Remarkably, TAC rapidly and progressively induced decompensation and dilation in Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice compared with no effect in any of the control groups (Figure 3B and Table 2). Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, but not controls, showed severe pulmonary edema following TAC (lung to body weight ratio: 20.45±0.71 mg/g in Gata4fl/flMHC-Cre; 18.51±0.70 mg/g in Gata4fl/flMHC-Cre; and 10.35±0.91 mg/g in controls [P<0.05]). Consistent with the total extent of GATA4 protein deletion in the myocardium, Gata4fl/flMHC-Cre showed a more severe profile of decompensation and dilation compared with Gata4fl/flMHC-Cre mice (Figure 3B and Table 2). Also of interest, all groups showed a similar surgical lethality profile (10%), although 3 of 23 Gata4fl/flMHC-Cre mice subjected to TAC died within the postsurgical period, whereas none of the other groups showed any lethality in this period (supplemental Table II). Collectively, these results indicate that GATA4 is required for compensation of heart performance following pressure overload stimulation.
Deletion of Gata4 Attenuates the Cardiac Hypertrophic Response
Although GATA4 has been implicated as a mediator of the cardiac hypertrophic response, a loss-of-function approach has yet to be evaluated in vivo. Here we examined the extent of cardiac hypertrophy by assessment of VW/BW and by measurement of cell areas in heart histological sections from TAC mice after 4 weeks of stimulation. Both Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice showed a significant 40% to 50% reduction in the hypertrophic response compared with each of the control groups subjected to TAC for 2 or 4 weeks (Figure 4A and data not shown). However, Gata4fl/flMHC-Cre TAC mice showed no enhancement in histopathology over wild types (data not shown), and fibrosis was not significantly greater (Figure 4B). At the cellular level, Gata4fl/flMHC-Cre mice showed a significant reduction in cardiomyocyte area following TAC for 4 weeks (Figure 4C). Interestingly, cardiac histological sections were also costained for GATA4 from Gata4fl/flMHC-Cre mice, and the 30% of residual cells with GATA4 protein expression were significantly larger after TAC compared with neighboring cells within the same heart that were GATA4-deficient (Figure 4C). These results suggest that GATA4 functions in a cell autonomous manner to regulate hypertrophy. Echocardiography-based assessment of ventricular wall thicknesses also showed attenuated growth in Gata4fl/flMHC-Cre mice compared with controls (Table 2). Mice were also subjected to Doppler echocardiography for analysis of transaortic pressure gradients developed across the constriction, which showed no differences between groups (data not shown). Lastly, Gata4fl/flMHC-Cre mice were also subjected to swimming to evaluate exercise-induced cardiac hypertrophy, which showed significantly less growth compared with wild-type and -MHC-Cre mice (Figure 4D). Collectively, these results indicate that GATA4 is a necessary mediator of the cardiac hypertrophic response in vivo.
Loss of Gata4 Increases Cell Death in the Heart
The observation that Gata4fl/flMHC-Cre mice showed baseline ventricular dilation and a progressive reduction in cardiac functional performance with age suggested that cell death might be involved. Indeed, Gata4+/eC mice were previously shown to have enhanced cell death in the heart following doxorubicin treatment.24 To determine whether enhanced cell death might partially underlie the cardiac phenotype described above, TUNEL was performed in adult and embryonic hearts from Gata4fl/flMHC-Cre and Gata4fl/flNkx2.5-Cre mice, respectively. As speculated, baseline TUNEL was significantly increased in Gata4fl/flMHC-Cre mice compared with wild-type and -MHC-Cre mice alone at 12 and 20 weeks of age (Figure 5A). Moreover, TAC stimulation for 4 weeks induced a greater increase in TUNEL in Gata4fl/flMHC-Cre mice compared with the control groups (P<0.05) (Figure 5A). These results suggest that loss of cardiac myocytes at baseline and following pressure overload stimulation contributes to the decompensation observed in Gata4fl/flMHC-Cre mice.
To more thoroughly evaluate the hypothesis that loss of GATA4 enhances cardiac TUNEL within another context, embryos from Gata4-loxPeCtargeted mice crossed with the Nkx2.5-Cre knock-in allele were examined at E12.5.19 The Nkx2.5-Cre allele produces much earlier expression of Cre in the heart, resulting in a greater than 95% loss of GATA4 protein from the myocardium by E12.5 of development (Figure 5B). Targeting of Gata4 in this manner resulted in embryonic lethality between E12.5 and E15.5, with a uniform reduction in ventricular wall thicknesses (data not shown). However, a more extensive description of the embryonic phenotype is not presented here because it has been given by 2 other groups.14eC16 Consistent with the observations made in the adult heart, Gata4fl/flNkx2.5-Cre embryos showed significantly higher rates of TUNEL in the heart compared with Gata4fl/fl or Gata4fl/flMHC-Cre embryos, the latter of which had not yet lost significant GATA4 protein expression (Figures 5C and 1F). Thus, loss of GATA4 from the adult or embryonic heart likely enhances myocyte apoptosis levels.
Loss of Gata4 From the Heart Dramatically Alters Basal Gene Expression
Although cellular apoptosis may partially explain the mechanistic underpinnings of decompensation and lethality in Gata4 heart-targeted mice, the exact molecular mechanism was uncertain. Because GATA4 is a transcription factor, the obvious hypothesis is that a subset of cardiac-expressed genes involved in cellular death and/or decompensation would be altered. This hypothesis was evaluated in an unbiased manner by array screening with the Affymetrix MOE 430 to 2 chip set using RNA from the hearts of 8-week-old Gata4fl/flMHC-Cre mice and 2 different control groups (Figure 6A). Eight weeks was selected for generation of RNA because this time largely precedes any pathological manifestations, so that secondary alterations in gene expression were less likely, and, at this time, there was no basal activation of select signaling kinases involved in cardiac stress stimulation (supplemental Figure). Compared with both control groups, hearts from Gata4fl/flMHC-Cre mice showed a significant change in 362 genes compared with cardiac RNA from wild-type mice, which is approximately 1.0% of all surveyed genes (Figure 6A). Most notably, GATA4 was previously shown to directly regulate expression of the ANF, -MHC, and -MHC genes, each of which is significantly altered in the hearts of Gata4fl/flMHC-Cre mice (Figure 6B). Interestingly, 3 genes with a known role in regulating apoptosis were also significantly altered in the absence of Gata4 (Figure 6B and 6C). For example, protein kinase C (PKC) expression was reduced in Gata4-deleted hearts, which based on previous literature, should enhance cell death.25 Similarly, Gata4-deleted hearts showed an upregulation in expression of the proapoptotic factor caspase-12 (csp12) and Bcl6. Gata4-deleted hearts also had significant alterations in expression of other signaling and transcriptional regulatory factors such as Sox7, Iroquois-related homeobox gene 3 (Irx3), fibroblast growth factor 1 (FGF1), and prostaglandin F receptor (PGFR) (Figure 6B and 6C). In conclusion, targeted deletion of Gata4 from the myocardium leads to profound alterations in cardiac gene expression, a subset of which might partially underlie the observed increase in myocyte TUNEL or ventricular decompensation and loss of hypertrophic potential.
Discussion
GATA4 has been ascribed to a number of critical functions in the heart, spanning from the specification and differentiation of cardiac myocytes early in development to the regulation of the cardiac hypertrophic response in the adult. GATA4 mediates these processes through the direct transcriptional control of key cardiac structural and regulatory genes.1 For example, GATA4 has been shown to directly bind the promoters of the ANF, BNP, -MHC, and -MHC genes, thereby controlling their expression in the heart.1 Indeed, 3 of these 4 genes showed altered expression in the hearts of Gata4fl/flMHC-Cre mice, validating the previously proposed proximal regulatory role of GATA4. Cardiac-specific deletion of Gata4 also resulted in the dysregulation of approximately 1.0% of all cardiac-expressed genes, further supporting the overall importance of GATA4 as a homeostatic regulator of gene expression in the heart.
GATA4 is also thought to function as a critical regulator of cardiac hypertrophy, although most studies conducted to date relied on cultured neonatal cardiac myocytes as a model system. For example, overexpression of GATA4 in culture by adenoviral gene transfer induced cardiomyocyte hypertrophy, indicating the sufficiency of GATA4 in this process.7 More significantly, expression of dominant negative GATA4 (engrailed fusion) or antisense GATA4 mRNA each blocked GATA4-directed transcriptional responses and features of cardiomyocyte hypertrophy induced by phenylephrine and endothelin-1 in culture.7,8 In vivo, mild overexpression of GATA4 in the mouse heart by transgenesis induced a progressive hypertrophic response.7 However, direct assessment of the necessity of GATA4 in mediating cardiac hypertrophy in vivo had not been examined. Here we demonstrated for the first time that reduction in GATA4 expression by 70% or 95%, specifically within cardiac myocytes, attenuated the cardiac hypertrophic response following 2 or 4 weeks of pressure overload or following exercise stimulation in Gata4fl/flMHC-Cre mice. Interestingly, the 95% reduction in total GATA4 positive cardiac myocytes induced by the -MHC-Cre transgene showed a more consistent reduction in cardiac hypertrophy and earlier functional decompensation (Figure 3A and 3B and Table 2) compared with a 70% reduction in GATA4 positive cardiac myocytes induced by the -MHC-Cre transgene, suggesting a dosage-dependent effect. It is also interesting to note that cardiac-specific deletion of Gata4 only reduced the cardiac hypertrophic response by approximately 40% to 50%, suggesting either that other regulators are sufficient to mediate some degree of cardiac hypertrophy in the absence of Gata4 or that Gata6 can partially compensate for the loss of Gata4 in the adult heart (Gata5 is not expressed in the adult heart). However, loss of GATA4 did not affect postnatal growth of the heart, also referred to as developmental hypertrophy, suggesting that different transcriptional regulators are involved in this process, or that GATA6 can compensate for this type of growth. With respect to functional decompensation after pressure overload, the phenotype of Gata4-deleted mice is reminiscent of melusin gene targeting, which is a muscle-specific 1-integrin interacting protein. Loss of melusin compromised the hypertrophic response and promoted functional decompensation, in contrast to the phenotype of Gq-inhibited transgenic mice or dopamine -hydroxylase gene-targeted mice, which had inhibited cardiac hypertrophy after pressure overload without functional decompensation.26,27
Traditional germline disruption of Gata4 in the entire mouse resulted in early embryonic lethality between E7.0 and E9.5 because of defects in endoderm and ventral morphogenesis.22,23 However, these embryos still generated cardiac tissue that expressed heart-specific structural genes, suggesting that GATA4 was not required for specification and efficient differentiation, although GATA5 and GATA6, which are each expressed in the early developing cardiac splanchnic mesoderm, likely compensated.22,23 More recently, 2 additional approaches were used to gain insight into the role of GATA4 in later aspects of cardiac development. Tetraploid embryo complementation was employed using Gata4eC/eC embryonic stem cells, which generated embryos that progressed further in development and showed hypoplastic ventricles and a loss of the proepicardium, resulting in lethality.14 Gata4 was also deleted specifically in the heart by Pu and colleagues using a Cre-loxP-based approach and the same Nkx2.5-Cre knock-in allele that we used.15 However, the loxP insertion sites selected by Pu et al produced a hypomorphic Gata4 allele that reduced basal expression by approximately 50%.15 These Gata4fl/fl mice showed early embryonic lethality when crossed into with the Nkx2.5-Cre allele, and later embryonic lethality when crossed with an -MHC-Cre transgene.15,16 In contrast, our Gata4-loxP targeted allele did not disrupt basal expression so that both Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice were viable and amenable to analysis as adults. However, consistent with Pu et al, Gata4fl/flNkx2.5-Cre mice showed embryonic lethality, but at a slightly later time point with our Gata4-loxPeCtargeting strategy (E12.5-E15.5). The reason underlying lethality in Gata4fl/flNkx2.5-Cre embryos, but not Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, is likely attributable to the much earlier profile of Nkx2.5-Cre expression compared with the -MHC and -MHC transgenes.
Mechanistically, adult hearts from Gata4fl/flMHC-Cre mice showed greater TUNEL at baseline, as well as significantly enhanced levels of TUNEL following pressure overload stimulation. Moreover, hearts from Gata4fl/flNkx2.5-Cre embryos at E12.5 also showed enhanced TUNEL compared with Gata4fl/flMHC-Cre embryos, which do not have appreciable GATA4 reductions until later in development. Thus, loss of Gata4 from the embryonic or adult heart predisposes to myocyte loss by apoptosis, likely contributing to the observed ventricular thinning in adults and hypoplastic ventricles in embryos. However, Pu and colleagues also reported a reduction in cellular proliferation in the absence of Gata4 in the developing heart, which could also play an important role.16 Our observations of increased TUNEL in the absence of Gata4 is consistent with the results of Aries et al, in which Gata4+/eC mice (heterozygotes) showed greater functional decompensation following doxorubicin treatment and greater TUNEL in cultured cardiomyocytes infected with an antisense mRNA expressing GATA4 adenovirus following doxorubicin treatment.24 By comparison, doxorubicin-mediated cell death in cultured cardiomyocytes or HL-1 cells was reversed by GATA4 or GATA6 overexpression.28 Moreover, depletion of GATA4 from chicken embryos enhanced cellular apoptosis in the heart-forming region and underlying endoderm.29 Collectively, these results suggest that GATA4 regulates, in part, cellular viability at baseline and in response to stress stimulation.
However, it is uncertain whether cellular attrition through apoptosis directly underlies the progressive decompensation that was observed in Gata4fl/flMHC-Cre and Gata4fl/flMHC-Cre mice, as we were unable to directly quantify total cell number in adult hearts of the various genotypes examined here. It is also formally possible that many of the profound alterations in gene expression that occurs in the absence of Gata4 could underlie the propensity toward decompensation without a loss in myocyte number. Indeed, the dramatic shift from -MHC to -MHC expression, as observed in the hearts of Gata4fl/flMHC-Cre mice, would be predicted to reduce the contractile performance of the myocardium, hence leading possibly to greater decompensation after stress stimulation. However, it is likely that a progressive loss of cardiac myocytes, together with the observed alterations in cardiac structural and regulatory gene expression, leads to the observed phenotype in cardiac-specific Gata4-deleted mice.
Acknowledgments
This work was supported by grants from the NIH, an American Heart Association Established Investigator Grant (to J.D.M), and an American Heart Association Postdoctoral Fellowship grant (0425393B to T.O.).
References
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