Published online before print March 3, 2008, doi: 10.1161/CIRCULATIONAHA.107.759233
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(Circulation. 2008;117:1414-1422.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Heart-Specific Ablation of Prkar1a Causes Failure of Heart Development and Myxomagenesis
From the Department of Molecular Virology, Immunology, and Molecular Genetics (Z.Y., G.N.J., W.H.T., L.S.K.), Center for Biostatistics (X.Z., D.J.), Division of Cardiology (P.F.B.), and Division of Endocrinology, Diabetes, and Metabolism (L.S.K.), Ohio State University, Columbus, and Division of Endocrinology Metabolism and Diabetes and Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City (E.D.A.).
Correspondence to Lawrence S. Kirschner, 420 W 12th Ave, TMRF 544, Columbus, OH 43210. E-mail Lawrence.Kirschner{at}osumc.edu
Received January 16, 2007; accepted January 23, 2008.
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Abstract |
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Background— Protein kinase A signaling has long been known to play an important role in cardiac function. Dysregulation of the protein kinase A system, caused by mutation of the protein kinase A regulatory subunit gene PRKAR1A, causes the inherited tumor syndrome Carney complex, which includes cardiac myxomas as one of its cardinal features. Mouse models of this genetic defect have been unsatisfactory because homozygote null animals die early in development and heterozygotes do not exhibit a cardiac phenotype.
Methods and Results— To study the cardiac-specific effects resulting from complete loss of Prkar1a, we used cre-lox technology to generate mice lacking this protein specifically in cardiomyocytes. Conditional knockout mice died at day 11.5 to 12.5 of embryogenesis with thin-walled, dilated hearts. These hearts showed elevated protein kinase A activity and decreased cardiomyocyte proliferation before demise. Analysis of the expression of transcription factors required for cardiogenesis revealed downregulation of key cardiac transcription factors such as the serum response factor, Gata4, and Nkx2–5. Although heart wall thickness was reduced overall, specific areas exhibited morphological changes consistent with myxomatous degeneration in the walls of knockout hearts.
Conclusions— Loss of Prkar1a from the heart causes a failure of proper myocardial development with subsequent cardiac failure and embryonic demise. These changes appear to be due to suppression of cardiac-specific transcription by increased protein kinase A activity. These biochemical changes lead to myxoma-like changes, indicating that these mice may be a good model with which to study the formation of these tumors.
Key Words: cardiomyopathy • cyclic AMP-dependent protein kinases • genes • myxoma • signal transduction
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Introduction |
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The cAMP-dependent protein kinase (protein kinase A [PKA]) signaling system is one of the central means by which cardiac function is regulated in the body. PKA activation is achieved by stimulation of 7-transmembrane G-protein–coupled receptors, which couple to adenylate cyclase and produce cAMP. In the heart, cAMP production is classically medicated by the β1- and β2-adrenergic receptors,1 although other receptors also use this same second messenger system.2 Once generated, intracellular cAMP binds to the regulatory subunits of PKA, releasing the catalytic subunit, which phosphorylates proteins involved in contractile function, including key targets such as the ryanodine receptor 2 (Ryr2) and the sarcoplasmic Ca2+-ATPase. These effects modulate the actions of catecholamines on cardiac function. Upregulation of PKA activity has been posited to occur in the failing heart because of increased phosphorylation of PKA targets,3,4 providing an explanation for the beneficial effects of β-blockade in these patients.
Clinical Perspective p 1422
From a genetic point of view, dysregulation of the PKA system has been associated with abnormalities of heart function in mouse models,1 although corresponding human syndromes are generally unknown. The singular example of a PKA abnormality causing an inherited human syndrome affecting the heart is the Carney complex (CNC). This genetic disease is caused by inactivating mutations of PRKAR1A, which encodes the type 1A regulatory subunit of the PKA holoenzyme.5,6 Although the phenotype includes skin pigmentation and endocrine tumors, a predisposition for the development of cardiac myxomas is one of its cardinal features. Approximately 30% to 60% of CNC patients will develop cardiac myxomas,7 and cardiac complications are the cause of death in more than half of the them.8 In tumors from CNC patients, PKA activity is elevated,5 a change that also is observed in mouse cells engineered to lack the Prkar1a protein.9
To model CNC, Prkar1a knockout mice have been generated.10,11 Although homozygous null mice die early in development, heterozygotes are tumor prone11,12 with a propensity to develop neoplasms in cAMP-responsive tissues.11 To date, no abnormalities of cardiac function have been described,12 and myxomas have not been observed.
To better examine the effects of ablation of Prkar1a on cardiac development and function, we have undertaken the generation of mice lacking this protein in the heart. Using a cre-lox approach, we produced a cardiac-specific knockout (CKO) of Prkar1a, which has allowed us to avoid the general failure of mesenchymal development observed in the complete knockout.10 In the present report, we show that Prkar1a-CKO mice die around day 11.5 of embryogenesis (e11.5) as a result of a thinned and dilated myocardium. At the cellular level, we see a failure of cardiomyocyte proliferation and downregulation of cardiac-specific transcription factors. Intriguingly, we also demonstrate that cells in these failing hearts undergo myxomatous degenerative changes, providing new potential insights into the formation of these poorly understood tumors.
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Methods |
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Animals
All animal studies in this report were conducted under the highest standards of animal care and were carried out under an Institutional Laboratory Animal Care and Use Committee–approved animal protocol. Prkar1a conditional null11 and
-myosin heavy chain (MHC)-Cre13 mouse lines and genotyping conditions have previously been described. Note that MHC has been designated as Myh6, but the mouse line is referred to as MHC-Cre for consistency.
Histology, Immunohistochemistry, and Whole-Mount X-Gal Staining
Staged embryos were processed and stained with hematoxylin and eosin for general morphology. Antibodies to MHC were from Abcam (Cambridge, Mass). Secondary antisera and horseradish peroxidase conjugates were from Vector Laboratories (Burlingame, Calif). Images were acquired with an Olympus (Melville, NY) digital camera and processed in Photoshop 7.0 (Adobe Systems, San Jose, Calif). All quantitative data were analyzed with MetaVue software (Molecular Devices, Union City, Calif). For cre reporter crosses, β-galactosidase activity was visualized by whole-mount X-gal staining at 37°C for 7 hours.
For analysis of cardiac structural parameters, MetaVue software was used to perform quantitative measurements. The atrial area was calculated as the area bounded by the right and left atrial walls and the endocardial cushion. The compact layer thickness was determined as the linear distance spanning the morphologically identified compact layer of the cardiac ventricle. For each heart, 10 measurements were taken at different locations in the ventricle (including both left and right ventricles) and averaged. The trabecular area was determined from the immunostained hearts by calculating the total area of brown coloration in the region below the endocardial cushion and bounded by the inner (endocardial) side of the compact layer. Seven and 6 control and CKO hearts, respectively, were used for all calculations. For quantitative analysis of myosin staining, 3 pairs of samples were analyzed for threshold values from control, and CKO slides were prepared and stained in parallel.
Cell Proliferation and Apoptosis
To determine cell proliferation rates in embryos, pregnant females were given intraperitoneal injections of 100 µg/g body weight of 5'-bromo-2'-deoxyuridine (BrdU; Sigma, St Louis, Mo) 2 hours before death. Proliferating cells were detected with an anti-BrdU antibody (BD Biosciences, San Jose, Calif) according to the manufacturer’s instructions. The proliferative index was determined as the ratio of BrdU-positive nuclei to total nuclei. Cell death was detected with a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay kit (Roche Applied Science, Indianapolis, Ind) or immunostaining for cleaved caspase-3 (Cell Signaling Technology, Danvers, Mass).
Quantitative Reverse-Transcription Polymerase Chain Reaction
Total RNA was isolated from e11.5 mouse hearts with the Qiagen RNeasy kit (Qiagen, Valencia, Calif), and cDNA was synthesized with an iScript kit (BioRad, Hercules, Calif). Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed with iQ SYBR green Supermix reagents and an iCycler (Bio-Rad Laboratories, Hercules, Calif). Primers for target genes were designed with the Primer3 software package14 (http://frodo.wi.mit.edu/primer3/input.htm) to cross exon boundaries. Primer sequences used in this study are given in Table I of the online-only Data Supplement. All PCRs were performed in triplicate, and mRNA fold changes were calculated by the 
Ct method using GAPDH as a standard.
Electron Microscopy
Thin sections (70 nm) from e11.5 embryos were processed for electron microscopy and examined with a Philips 301 transmission electron microscope (Eindhoven, the Netherlands). Images were captured with a Megaview III digital camera (Soft Imaging System, Lakewood, Colo).
Western Blot and Kinase Assays
Embryonic hearts were isolated and pooled in ice-cold PBS and then homogenized in lysis buffer containing 10 mmol/L Tris-HCl, pH7.1, 1 mmol/L EDTA, 1 mmol/L DTT, 0.5% NP-40, 0.1 mmol/L vanadate, 1 mmol/L NaF, and Halt protease inhibitors (Pierce, Rockford, Ill). After centrifugation, the supernatants were stored at –70°C for measurement of PKA subunits and PKA. Proteins were resolved in SDS-PAGE gels and probed with monoclonal antibodies for Prkar1a, Prkar1b, Prkar2b, and Prkac (BD Biosciences). Quantification was undertaken with ImageJ software. Basal activity and total PKA activity were measured with a nonradioactive PKA assay as previously described.9
Statistical Analysis
The quantitative assays in Figures 2, 4, and 5
were analyzed by a 2-sided t test. For statistical analysis of quantitative RT-PCR data (Figure 6), individual knockout animals were analyzed in triplicate for each transcript. Mixed linear models were used for the analysis to take advantage of having knockout and control mice from the same litter. To provide strict type I error control at =0.05 across several genes, we used an spending approach across 3 categories of genes. We used =0.03 for 5 transcription factors (primary hypotheses), =0.01 for each of the 5 structural genes, and =0.01 for the 5 functional genes (secondary hypotheses). Within each category, we used Holm’s procedure15 to control for the multiplicity of comparisons. In other words, we used very strict error control so that anything declared significant cannot reasonably be explained by chance. We used 1-sided testing because of the clear expectations for all genes tested. A complete description of statistical procedures is included in the online-only Data Supplement, in a table of summary statistics (online-only Data Supplement Table II), and in the detailed results of the statistical analysis (online-only Data Supplement Table III).
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The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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CKO of Prkar1a Causes Early Embryonic Lethality Owing to Failure of Cardiac Development
To assess the effects of ablation of Prkar1a on cardiac function, we crossed the
MHC-Cre allele13 into Prkar1a conditional null animals to generate Prkar1a-CKO mice (MHC-Cre;Prkar1aloxP/loxP, henceforth called CKO). These matings did not result in the birth of CKO mice, although other genotypes were present at mendelian ratios and demonstrated no phenotypic abnormalities. Genotyping of embryos during gestation allowed recovery of mutant embryos at the expected ratios between e9.5 and e11.5; however, no viable mutant embryos were observed past e12.5. At e11.5, the CKO embryos were discernible grossly by their pale appearance, smaller size, and particularly the dilated pericardial lumen (Figure 1). The hearts of the mutants retained rhythmic contraction but at a slower rate than control littermates. The mutant hearts displayed an enlarged right atrium, and an abnormal accumulation of a white gelatinous material was noted between the left atrium and left ventricle that caused a subtle but clear distortion of the cardiac anatomy (Figure 1C and 1D). This abnormality is better appreciated at the histopathological level, as described below.
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Histological analysis revealed no changes in the CKO hearts at e9.5 (data not shown). However, as early as e10.5 (data not shown) and especially at e11.5, marked changes were observed in cardiac morphology (Figure 2A and 2B). To better define the cardiac anatomy, specimens were immunostained for MHC (Figure 2C and 2D). This analysis also revealed that the mutant hearts exhibited a marked thinning of the ventricular walls and atrial dilation (Figure 2E). In the left ventricle, CKO mice demonstrated a global reduction in cardiomyocytes, as evidenced by both a dramatically thinned compact layer (Figure 2F) and poorly developed trabeculation (Figure 2G) compared with wild-type (WT) controls.
Ultrastructural analysis of cardiac fibers by electron microscopy (Figure 3) revealed that CKO hearts (Figure 3B) exhibited disorganized sarcomeres. Specifically, the regular appearance of the Z disks was disrupted compared with the WT hearts (Figure 3A). Myofilaments were disordered and appeared less dense, and Z disks appeared disconnected from the remainder of the sarcomere. In addition, an obvious defect was observed in sarcomeric structures, with the mutant cells lacking the I band and M line (Figure 3B).
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To understand the basis for the dramatic reduction in cardiomyocytes in the mutants, we studied markers of proliferation and apoptosis. The proliferation rate was determined by measuring BrdU incorporation into cardiomyocytes. Because hearts from mice at e11.5 already showed signs of degeneration, we studied proliferative rates from both e10.5 and e11.5 embryos (Figure 4A through 4D). We observed a significant reduction in the proliferation index in mutant hearts at both ages (Figure 4E and 4F). To test for excess apoptotic cell death, we performed terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling and immunostaining for cleaved caspase-3 on control and CKO hearts at e11.5. Surprisingly, <1% of myocardial cells exhibited apoptotic changes in either genotype, whereas abundant apoptotic cells were observed in appropriate areas elsewhere in the mice (data not shown).
Alterations of PKA Isoforms and Increased PKA Activity in CKO Hearts
Next, we undertook to determine the effect of this genetic manipulation on the PKA system as a whole. In CKO hearts, Prkar1a protein was reduced by 
61% (Figure 5A and 5B), whereas the levels of other PKA regulatory or catalytic subunits were not significantly altered. This observation is in contrast to observations made in other PKA regulatory subunit knockout models in which upregulation of Prkar1a typically is observed.16 Because residual Prkar1a in the CKO hearts was higher than anticipated, we investigated the efficiency of Cre-mediated recombination in this model. By crossing the MHC-Cre mice with the Rosa26lacZ reporter line,17 we determined that 55% of cells within the myocardium had undergone recombination (online-only Data Supplement Figure II), suggesting complete ablation of Prkar1a in cardiomyocytes expressing Cre.
We measured PKA activity in CKO and control hearts at e11.5 to assess the functional effects of Prkar1a ablation. Proteins from 3 to 5 hearts for each condition were pooled and used together to measure PKA activity. As shown in Figure 5C, mutant hearts exhibited elevated PKA activity as both free PKA activity (without exogenous cAMP) and total PKA activity (with exogenous cAMP). These alterations are consistent with previous observations on Prkar1a-null cells in tissue culture9 or from tumors.5,18
Prkar1a-CKO Embryos Demonstrate an Impaired Expression of Critical Cardiac Genes
Given the phenotype of Prkar1a-CKO embryos at the microscopic and ultrastructural levels, we performed quantitative RT-PCR to examine whether the loss of Prkar1a, with resultant PKA dysregulation, led to altered expression of cardiac-specific genes (Figure 6). As proof of principle, we first examined expression of Prkar1a and the PKA target gene Fos.19 This analysis illustrated that Prkar1a expression showed a 62% reduction (–Ct of –1.39±0.31; P=0.0004), in good agreement with the estimated frequency of Cre-mediated recombination in CKO hearts (supplementary Figure I). In accordance with their elevated PKA activity, CKO hearts demonstrated 4.7-fold elevated levels of Fos transcripts (–Ct of 2.23±0.42; P=0.0001). Next, we probed the expression of transcription factors important for cardiac development (Srf, Nkx2–5, Gata4, Tead1, Myocd), which we expected to go down on the basis of a comparison of the Prkar1a-CKO phenotype and other knockout models.20–23 With the exception of Tead1, all of the factors involved in cardiac transcription were significantly downregulated according to our strict significance criteria (Tead1 also was lower but not significantly). Residual transcript levels were between 34% and 46% lower than the WT controls. Finally, we examined transcript levels of cardiac structural proteins such as members of the actin and myosin families (Myh6 [MHC], Myh7 [βMHC], Acta1, Acta2, Actc1) and other cardiac-specific functional proteins that may be influenced by PKA activity, including those involved in calcium handling (Slc8a1 [Ncx1], Ryr2), contraction (Tagln [SM22] and Cnn1), and other functions (Nppa [Anf}). Although all genes were observed to go down as expected, only Acta2 (aortic smooth muscle actin) and Nppa (natriuretic peptide precursor type A) were observed to meet the predetermined strict criteria for significant changes (see the online-only Data Supplement). The reduction in expression of these transcripts was 40%, which was quite similar to the reductions in the transcriptions controlling them. Although not significant, the expression levels for other genes were reduced by 35±2% on average.
To verify that the reductions in message correlated with reduced protein expression, we performed Western blotting for Srf; this analysis confirmed a marked reduction in this transcription factor in the CKO hearts (supplemental Figure II). To investigate whether the reductions in the Myh7 message (52% reduction; P=0.0105) correlated with reduced protein expression, we quantified the density of immunohistochemical staining for myosin in the heart sections. Analysis of 3 CKO and 3 control mice demonstrated a 60% reduction in myosin staining in the knockout animals (Figure 2C and 2D), which matched well to the measured transcript level.
Myxoma-Like Lesions and Cytological Atypia Are Present in Prkar1a-CKO Mouse Hearts
During our analysis of Prkar1a-CKO hearts, we noted that the large majority of mutant hearts exhibited focal areas of thickening in the cardiac walls and abnormalities of the septa (compare Figure 7A through 7C). The lesions were located most commonly between the left atrium and left ventricle and corresponded to the white gelatinous material observed grossly in the mutant hearts (Figure 1D). Histologically, these lesions comprised loose myxoid stroma and/or gland-like tissue (Figure 7B, 7C, 7E, and 7F). Within this stroma were single cells or clusters of round, polygonal, or stellate cells scattered in the loose myxoid stroma under the endocardial or epicardial lining (Figure 7E). The tissue was rich in acid and neutral mucopolysaccharides (as evidenced by staining with Alcian blue and periodic acid–Schiff; Figure 7G and 7H), a staining pattern typical of human cardiac myxomas. Intriguingly, the gland-like tissue appeared to produce neutral mucins with evidence of strong intracellular periodic acid–Schiff staining (Figure 7I). This histological change has been reported in human myxomas at a rate of 3%.24,25
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In addition to this histological appearance, we also noted areas of cellular atypia in CKO hearts (Figure 8). In these areas, the tissue lost its normal organization, and the cells did not show polarization. Moreover, the cells exhibited unequivocal cytological features of early malignant changes with cellular and nuclear pleomorphism, indistinct cell borders, high nuclear-to-cytoplasm ratios, and an elevated mitotic index.
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Overall, 24 mutant mice identified from 10 litters were analyzed at the histopathological level for these morphological changes. Twelve of 24 mice (50%) exhibited myxomatous changes in the heart, whereas 17 of 24 mice (70.8%) exhibited cellular atypia. Eight of the 24 mutant mice (33%) exhibited both types of pathological change, suggesting that these 2 phenotypes are related to the same underlying molecular pathology.
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PKA in Cardiac Development
The PKA system has long been known to play a key role in cardiac function and has been studied in mice by the use of cardiac-targeted transgenes such as β-adrenergic receptors or the PKA-C subunit.1 However, a role in cardiac development has not previously been appreciated.
Here, we present clear evidence that proper regulation of PKA during development is required for normal cardiac development. In mice lacking Prkar1a, embryos die before e9.5 because of developmental failure of mesodermal derivatives.10 Heart tubes are not formed, although cardiomyocytes are identifiable in the mutants. In contrast, mice lacking Prkar1a only in their hearts developed normal cardiac structures, with clearly identifiable atria, ventricles, and pericardial structures (Figure 1). However, cardiac failure became evident at e11.5 with marked thinning of ventricular walls characterized by both hypoplasia of the compact layer and severely reduced trabeculation.
The phenotype of embryonic demise resulting from a thinning of the myocardium has previously been observed on inactivation of the transcription factors Srf20,22 and Gata4.21,23 Ultrastructurally, Srf mutant mice exhibit disordered sarcomeres, as was observed here.20 In Srf knockout hearts, increased apoptosis caused the decrease in cell number, whereas in our model, the defect appears to lie in reduced proliferation. This observation is likely due to a direct antiproliferative effect of PKA because the ability of PKA activation to inhibit cell proliferation has been well described in many cell systems, including cultured smooth muscle myocytes.26
Our finding of embryonic lethality contrasts with other genetic models for PKA activation, including transgenic overexpression of the catalytic subunit.27 Although the lack of embryonic lethality in this latter model may be due to the use of an Myh6 (MHC) promoter with only modest activity in the embryo, the reason for this discrepancy may lie in the regulation of PKA itself. In models in which other PKA regulatory subunits are knocked out, the Prkar1a subunit is upregulated as a means to control excess PKA activity.10 When Prkar1a itself is ablated, however, other subunits are not able to compensate, as shown by a lack of upregulation of other regulatory subunits in our model (Figure 5).
Although formation of the gross cardiac structures occurred, one of the striking aspects of the cardiac phenotype reported here was the disorganization of the sarcomeres in the Prkar1a-CKO hearts. Z disks were easily seen, but they were not properly assembled, and myofilaments appeared loose and disarrayed. Defective expression of Z-disk proteins like actin (Acta1 and Acta2) on the depletion of Prkar1a likely underlies this ultrastructural defect. In addition, the significantly diminished expression of actins and Myh7 (β-MHC) may result in a deficiency in both thin (actin) and thick (myosin) myofilament components and disruption of both M lines and I bands. Most likely, the disorganized Z disks and myofilaments lead to poor cardiac contraction and heart failure, causing demise of the growing embryo.
Mechanistically, ablation of Prkar1a caused a reduced transcription of genes required for cardiac development. These effects may be due to a direct effect of excess PKA activity on gene transcription or may be an indirect effect on other transcription factors because PKA has previously been observed to interfere with Srf activity.28,29 Given the significant reduction in the expression of key cardiac transcription factors such as Gata4, Srf, and Nkx2–5, we speculate that both direct and indirect effects likely play a role in the observed phenotype. The fact that transcription factors function in combination and in hierarchical networks30,31 may explain why a reduction in multiple proteins, as observed here, causes synergistic and deleterious effects during cardiogenesis. This hypothesis is corroborated by our data; reductions in both Gata4 and Nkx2–5 lead to significant reductions in Nppa, which requires these factors to work in concert for normal expression.30 In addition, we observed consistent downregulation of the Slc8a1 (Ncx1) message, although the reduction did not meet our strict criteria for significance (P=0.0127). This protein is required for Ca2+ extrusion32,33 and plays a pivotal role in the regulation of intracellular Ca2+ homeostasis in cardiac myocytes. Downregulation of this protein and consequent increases in cytosolic and sarcoplasmic Ca2+ can lead to Ca2+ overload–induced loss of mitochondrial membrane potential.34 This may contribute to both reduced cardiac function and arrhythmogenesis associated with heart failure in mouse models35 and human patients.
PKA in Myxoma Development
Most interestingly, 50% of Prkar1a-CKO hearts exhibit stroma-rich lesions suggestive of cardiac myxomas in human patients. At dissection, myxoid material typically was noted between the left atrium and left ventricle. This region of the heart is close to the left atrium, from which 75% cardiac myxomas originate in human patients.24,36 Histologically, the characteristic features of cardiac myxoma, including polygonal or stellate cells surrounded by abundant loose stroma rich in acid mucopolysaccharide, were observed in the majority of CKO hearts. Most strikingly, they also exhibit epithelium- or gland-like changes, which are rare but diagnostic features of human cardiac myxomas.24,25,37 Thus, the evidence is convincing that this mouse model develops early phases in the development of cardiac myxoma. With regard to the histogenesis of myxomas, most authors favor that myxomas arise from the endocardium38 and are derived from subendocardial multipotential mesenchymal cells.39,40 The Prkar1a-CKO mice displayed myxomatous lesions under both endocardial and epicardial cell linings. However, no cre-positive (ie, Prkar1a null) cells were observed within the endocardium or epicardium at e11.5 (supplemental Figure I). This finding suggests that cardiac myxomas may arise from mesenchymal cardiomyocyte progenitor cells. Alternatively, cardiomyocytes with depletion of Prkar1a might promote endocardial or epicardial cells to undergo tumorigenesis through a paracrine effect.11 Future lineage tracking experiments may help to resolve this issue.
To date, these findings represent the only model of myxomagenesis in the mouse. Myxomas also are seen in human patients associated with mutations in the perinatal myosin gene MYH8,41 but no mouse model of this condition has yet been described. Myxomas also are observed in rabbits infected with rabbit myxomas virus. In this instance, the virus is thought to interfere with immune function, but the connection between this physiological abnormality and tumorigenesis is as yet unknown.42 As is the case in our model, disruption of cell-cell signaling among different cardiac layers may promote myxomatous changes.
Relationship of the Mouse Model to Human Disease
Patients with CNC are heterozygotes for nonfunctional PRKAR1A alleles,5 and it is unclear how loss of the remaining allele affects tumor formation. Loss of the normal allele (ie, loss of heterozygosity) at the PRKAR1A locus has been observed in a subset of CNC-associated tumors5 and is observed in sporadic tumors of the adrenal and thyroid glands.18,43 However, loss of heterozygosity is not uniform, which has suggested to some authors that haploinsufficiency may be sufficient to promote tumorigenesis.12 In mice, the story is similar; some studies have detected allelic loss,11 but others have not.12 In the present study, all deleterious effects associated with Prkar1a mutations are caused by complete loss of the gene because they are not observed in conventional10–12 or conditional heterozygotes (data not shown). These observations include the striking finding of myxomatous degeneration in the walls of CKO hearts. Whether this observation in mice mimics the situation in humans remains unclear because many human tumor suppressor genes produce tumors in mice only in the null state.
Conclusions
In this study, we confirm that Prkar1a is required for cardiogenesis through a CKO of the gene. Prkar1a-CKO mutant embryos die between e11.5 and e12.5 as a result of heart failure caused by a thinned and disordered myocardium. The depletion of Prkar1a causes excess PKA activity, with resultant downregulation of the transcriptional activity of cardiac transcription factors and their downstream targets, including key cardiac structural proteins and proteins involved in calcium handling. These alterations cause reduced cardiac function and arrhythmogenesis, both of which may contribute to eventual heart failure. Intriguingly, Prkar1a-CKO mutants developed atypia and myxoma-like changes in the myocardium. Neither of these findings has been reported in conventional Prkar1a knockouts or in any other genetically modified mouse, suggesting that Prkar1a-CKO mice may serve as the first good model with which to study the formation of cardiac myxomas.
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Acknowledgments |
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We would like to thank Lisa Rawahneh for excellent technical assistance with the histological analyses described in this article.
Sources of Funding
This work was funded in part by National Institutes of Health grants HD01323 and CA112268 (to Dr Kirschner) and by grant CA16058 (to the Ohio State University Comprehensive Cancer Center).
Disclosures
None.
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The online-only Data Supplement, which contains tables and figures, can be found with this article at http://circ.ahajournals.org/cgi/content/ full/CIRCULATIONAHA.107.759233/DC1.
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