Prevention of cardiac hypertrophy in mice by calcineurin inhibition.

Hypertrophic cardiomyopathy (HCM) is an inherited form of heart disease that affects 1 in 500 individuals. Here it is shown that calcineurin, a calcium-regulated phosphatase, plays a critical role in the pathogenesis of HCM. Administration of the calcineurin inhibitors cyclosporin and FK506 prevented disease in mice that were genetically predisposed to develop HCM as a result of aberrant expression of tropomodulin, myosin light chain-2, or fetal beta-tropomyosin in the heart. Cyclosporin had a similar effect in a rat model of pressure-overload hypertrophy. These results suggest that calcineurin inhibitors merit investigation as potential therapeutics for certain forms of human heart disease.

Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain.

We have used an in vitro assay to identify sources of retinoic acid (RA) and transgenic mice to identify target domains in the developing forebrain. RA participates in a sequence of events that leads to the establishment of the olfactory pathway. First, the lateral cranial mesoderm activates an RA-inducible transgene in neuroepithelial cells in the olfactory placode and the ventrolateral forebrain. Then, neurons and neurites begin to differentiate in these two regions. Finally, olfactory axons grow specifically into the ventrolateral forebrain and subsequently are limited to the olfactory bulb rudiment. The coordination of these events, perhaps by common signals, implies that retinoid induction and retinoid-activated region-specific transcriptional regulation may help to define a forebrain subdivision and the peripheral neurons that provide its primary innervation.

Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice.

Retinoids play a critical role in cardiac morphogenesis. To examine the effects of excessive retinoid signaling on myocardial development, transgenic mice that overexpress a constitutively active retinoic acid receptor (RAR) controlled by either the alpha- or beta-myosin heavy chain (MyHC) promoter were generated. Animals carrying the alpha-MyHC-RAR transgene expressed RARs in embryonic atria and in adult atria and ventricles, but developed no signs of either malformations or disease. In contrast, beta-MyHC-RAR animals, where expression was activated in fetal ventricles, developed a dilated cardiomyopathy that varied in severity with transgene copy number. Characteristic postmortem lesions included biventricular chamber dilation and left atrial thrombosis; the incidence and severity of these lesions increased with increasing copy number. Transcript analyses showed that molecular markers of hypertrophy, alpha-skeletal actin, atrial natriuretic factor and beta-MyHC, were upregulated. Cardiac performance of transgenic hearts was evaluated using the isolated perfused working heart model as well as in vivo, by transthoracic M-mode echocardiography. Both analyses showed moderate to severe impairment of left ventricular function and reduced cardiac contractility. Thus, expression of a constitutively active RAR in developing atria and/ or in postnatal ventricles is relatively benign, while ventricular expression during gestation can lead to significant cardiac dysfunction.

The Ron/STK receptor tyrosine kinase is essential for peri-implantation development in the mouse.

The Ron/STK receptor tyrosine kinase is a member of the c-Met family of receptors and is activated by hepatocyte growth factor-like protein (HGFL). Ron activation results in a variety of cellular responses in vitro, such as activation of macrophages, proliferation, migration, and invasion, suggesting a broad biologic role in vivo. Nevertheless, HGFL-deficient mice grow to adulthood with few appreciable phenotypic abnormalities. We report here that in striking contrast to the loss of its only known ligand, complete loss of Ron leads to early embryonic death. Embryos that are devoid of Ron (Ron-/-) are viable through the blastocyst stage of development but fail to survive past the peri-implantation period. In situ hybridization analysis demonstrates that Ron is expressed in the trophectoderm at embryonic day (E) 3.5 and is maintained in extraembryonic tissue through E7.5, compatible with an essential function at this stage of development. Hemizygous mice (Ron+/-) grow to adulthood; however, these mice are highly susceptible to endotoxic shock and appear to be compromised in their ability to downregulate nitric oxide production. These results demonstrate a novel role for Ron in early mouse development and suggest that Ron plays a limiting role in the inflammatory response.

High-molecular-weight human epidermal transglutaminase.

Human stratum corneum was extracted in Tris-HCl containing EDTA and phenylmethylsulfonyl fluoride, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transblotted to nitrocellulose papers and reacted with rabbit antihuman epidermal transglutaminase (ETG) antibody. Protein-bound antibody was detected with a multistep peroxidase procedure. Proteins with a molecular weight of 50,000 (50kDa) and 72,000 daltons (72kDa) were stained when anti-ETG was used and not when second antibody alone or sera from nonimmunized animals were used. When ETG was treated with trypsin or organic solvents, there was no alteration in the mobility of the 50kDa ETG band, but there was complete disappearance of the 72kDa band. Antibody that bound 72kDa protein, when eluted from the blot, reacted with both 50kDa and 72kDa proteins; similarly, antibody that bound to the 50kDa protein, when eluted from the blot, reacted with both the 50kDa and 72kDa proteins. Partially purified 72kDa ETG activity was increased (3 to 16 times control levels) after heating at 56 degrees C in the presence of calcium and dithiothreitol or by treatment with trypsin. These studies, in conjunction with the previous studies of ETG activation, are consistent with there being two forms of ETG. The different forms may play a role in regulating enzyme activity.

Monoclonal antibodies to two different epitopes in a 30-kD CNBr peptide of the K1 and K2 keratins.

Two anti-keratin monoclonal antibodies, Kab-2 and Kab-3, with specificities for different epitopes of type II (basic) human epidermal keratins, were produced. These antibodies had different immunofluorescent staining patterns on human fetal epidermis. Western blots and solid phase RIA showed both antibodies bound to 65-67-kD basic keratins (K1 and K2) extracted from foreskin epidermis. Competitive binding studies with the two Kab antibodies and other anti-keratin monoclonal antibodies showed that Kab-2 and Kab-3 recognized related epitopes, distinct from the epitopes recognized by other anti-keratin antibodies AE-1, 2, and 3. Kab-2 and Kab-3 epitopes were distinguished by differences in their reactivity with peptides generated by Staphylococcus aureus V8 protease digestion of the K1 keratin; the antibodies recognized both common and unique peptides. Western blots of cyanogen bromide digests of the K1 keratin showed that both Kab antibodies reacted with a 30-kD fragment of the molecule presumed to be the N-terminal CNBr peptide. We interpret these data to indicate that in tissues, portions of the N-terminal region of the K1 keratin are differentially available for reaction with these monoclonal antibodies and that morphologic differences in staining with monoclonal antibodies to the same molecule can reflect epitope specificity or epitope availability related to supramolecular organization.

Reduced blood pressure and increased sensitivity of the vasculature to parathyroid hormone-related protein (PTHrP) in transgenic mice overexpressing the PTH/PTHrP receptor in vascular smooth muscle.

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is postulated to exert vasorelaxant properties by activation of the PTH/PTHrP type 1 receptor. As a model for studying the actions of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress the PTH/PTHrP receptor (PTHrP-R) in smooth muscle. Oocyte injection with a SMP8-PTHrP-R fusion construct yielded six founder mice. F1 offspring were viable and demonstrated selective overexpression of the SMP8-PTHP-R messenger RNA in smooth muscle-rich tissues. Baseline blood pressure measured in conscious mice by tail sphygmomanometry was significantly lower in the receptor-overexpressing mice than that in controls (117 +/- 4 vs. 133 +/- 3 mm Hg; P < 0.05). In anesthetized animals, iv infusion of PTHrP-(1-34)NH2 caused a significantly greater reduction in blood pressure and total peripheral resistance in transgenic mice than in control animals. Vascular contractility was studied in paired, isometrically mounted aortas from 9-week-old transgenic and wild-type mice. The force of contraction in response to phenlyephrine was not significantly different between transgenic and wild-type mice. However, PTHrP-(1-34) NH2 relaxed aortic vessel preparations from transgenic mice to a greater extent than in controls (77.1 +/- 3% vs. 38.4 +/- 4%; P < 0.001). To determine the impact of overexpression of PTH/PTHrP type 1 receptor and its ligand on the development of the cardiovascular system, double transgenic mice were created by crossing SMP8-PTHrP-R transgenic mice with mice overexpressing PTHrP (SMP8-PTHrP). Double transgenic mice died around day E9 with abnormalities in the developing heart. In conclusion, overexpression of PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure, probably through sustained activation of the receptor by endogenous ligand. The cardiovascular defects observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP may play a role in the normal development of the cardiovascular system.

The proximal promoter of the aldolase A gene remains active during myogenesis in vitro and muscle development in vivo.

The gene for aldolase A in mouse has been shown to be regulated by alternative promoters with attendant alternative first exons. The distal promoter/exon M functions only in muscle while the proximal promoter/exon H is active in early muscle development and in most other tissues. We have analyzed the developmental expression of M and H promoters in mouse throughout myogenesis both in vitro and in vivo. In C2C12 cells RNase protection assays revealed the M promoter is induced within 24 hr of the onset of myogenic differentiation, and both M- and H-specific mRNAs accumulate over 5 days in culture. Nuclear run-on transcription and in situ hybridization with an exon-specific probe demonstrate that the H promoter remains transcriptionally active even in differentiated myotubes. The in vitro results were then compared to similar RNase protection studies of M and H expression during muscle development in vivo. These data show a marked similarity between promoter activation and steady-state transcript accumulation in vivo and in vitro, but within a limited developmental time frame (E15 to 1 week postnatal). In situ hybridizations suggest that simultaneous transcription from both promoters may also occur early in muscle development. Furthermore, the M promoter shows no fiber-type restriction until 1 to 3 weeks postnatally, coincident with muscle maturation, while the H promoter remains transcriptionally active at all stages of development and in all fiber types.

Alternative promoter usage by aldolase A during in vitro myogenesis.

Aldolase A in the mouse, as in human and rat, shows tissue-specific variability of message size. In addition, in muscle tissue the mRNA size is also developmentally regulated. In order to determine whether this muscle-specific regulatory mechanism can be reproduced in vitro, we have examined the mRNA species of aldolase A isolated from mouse C2C12 myoblasts and myotubes on Northern blots and by primer extension. We show that aldolase A mRNA increases during in vitro myogenesis; that this induction is accompanied by a change in the message population; and that this change is due to activation of a muscle-specific alternative promoter.

Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus.

During fetal development, a specialized vessel the ductus arteriosus, shunts blood from the pulmonary artery to the aorta, thus bypassing the lungs. The ductus differs primarily from the great vessels in that it is a muscular rather than an elastic artery, and the etiology of this differential development remains controversial. We present evidence that retinoic acid (RA) may contribute to the unique muscle phenotype of the ductus arteriosus. Using a transgenic mouse carrying an RA response element-lacZ transgene that expresses beta-galactosidase (beta-gal) in response to endogenous RA signals during embryonic and fetal development, we observe a strong beta-gal signal in the ductus arteriosus. By immunofluorescence, this signal colocalizes with the expression of the adult-specific smooth muscle myosin heavy chain isoform, SM2. The beta-gal signal is present throughout fetal development and persists in the neonate until the ductus arteriosus is completely closed. beta-Gal-positive cells are first detected by immunofluorescence at 13.5 days postcoitum (dpc) in the mesenchyme surrounding the ductus. By 15.5 dpc, very intense beta-gal staining localizes to the ductus arteriosus but is absent or minimal in the pulmonary trunk and aortic arch; by 17.5 dpc, the smooth muscle layers of the tunica media in the ductus arteriosus exhibit positive beta-gal staining. Immunostaining with antibodies against smooth muscle myosins shows that, while SM1 is expressed in all embryonic vessels, SM2 is precociously expressed in the ductus arteriosus. Furthermore, SM2 expression can be detected in the ductus as early as 15.5 dpc. In the neonate, the beta-gal signal persists in the smooth muscle layer of the ductus and immunostaining colocalizes with SM2 expression. These data suggest that RA may play a role in inducing and maintaining smooth muscle differentiation in the developing ductus arteriosus and may promote precocious expression of the adult vascular phenotype.

Local sources of retinoic acid coincide with retinoid-mediated transgene activity during embryonic development.

We have assessed whether retinoic acid (RA) comes from local sources or is available widely to activate gene expression in embryos. We used an RA-responsive indicator cell line, L-C2A5, to localize RA sources. In these cells, an RA-sensitive promoter/lacZ reporter construct used previously by us to produce indicator transgenic mice is induced globally by RA in medium or locally by RA released at physiological concentrations (1 nM) from AG-1X2 resin beads. Furthermore, the cells are differentially responsive to the 9-cis and all-trans isomers of RA at low concentrations. Indicator transgenic mice with the same promoter/reporter construct were used to identify regions of RA-mediated gene activation. There are distinct domains of lacZ expression in the cervical and lumbar spinal cords of embryonic indicator mice. This pattern might reflect localized RA sources or restricted spatial and temporal expression of RA receptors, binding proteins, or other factors. To resolve this issue we compared the pattern of transgene activation in indicator cell monolayers cocultured with normal embryonic spinal cords with that in transgenic spinal cords. The explants induced reporter gene expression in L-C2A5 monolayers in a pattern identical to that in transgenic mice: alar regions of the cervical and lumbar cord were positive whereas those in the thoracic and sacral regions were not. We conclude that restricted sources of RA in the developing spinal cord mediate the local activation of RA-inducible genes. Thus, region-specific gene activation in embryos can be mediated by precisely localized sources of inductive molecules like RA.

Nonconservative utilization of aldolase A alternative promoters.

Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique arrangement of three tandem promoters and exons preceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their mature forms, differ only in the sequence of their 5'-untranslated regions. We have isolated and investigated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfection into C2C12 cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5'-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Consistent with previous reports, exon M is found only in skeletal muscle and exon H, the "housekeeping" exon, is utilized in every tissue where aldolase A is expressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction.

Retinoic acid inhibits cardiac neural crest migration by blocking c-Jun N-terminal kinase activation.

Retinoic acid (RA), a potent teratogen, produces a characteristic set of embryonic cardiovascular malformations similar to those observed in neural crest ablated avians. While the effects of RA on neural crest are well described, the molecular mechanism(s) of RA action on these cells is less clear. The present study examines the relationship between RA and mitogen-activated protein kinase signaling in neural crest cells and demonstrates that c-Jun N-terminal kinase (JNK) activation is severely repressed by RA. RA suppressed migration and proliferation of primary cultures of mouse neural crest cells treated in vitro as well as from animals treated in vivo. On Western blots, JNK activation/phosphorylation in neural crest cultures was reduced, while neither extracellular signal-regulated kinase (ERK) nor p38 pathways were affected. Both the dose-dependent stimulation of neural crest outgrowth and JNK phosphorylation by platelet-derived growth factor AA, which promotes outgrowth but not proliferation of neural crest cultures, were completely abrogated by RA. To establish the relevance of the JNK signaling pathway to cardiac neural crest migration, dominant negative adenoviral constructs were used to inhibit upstream activation of JNK or c-Jun downstream responses. Both adenoviral constructs markedly reduced neural crest cell outgrowth, while a dominant negative inhibitor of the p38 pathway had no effect. These data demonstrate that the JNK signaling pathway and c-Jun activation are critical for cardiac neural crest outgrowth and are potential targets for the action of RA.

Retinoid signaling and the generation of regional and cellular diversity in the embryonic mouse spinal cord.

Retinoid-dependent gene expression accompanies the emergence of distinct regions and cell classes in the mouse spinal cord around midgestation. We asked whether changes in the expression of retinoid signaling molecules and retinoid-responsive genes reflect the establishment of this regional and cellular diversity. At E10.5, retinoic acid (RA) receptors (RAR)alpha, RAR beta, the retinoid X receptor (RXR) gamma, cellular RA binding protein (CRABP)I, CRAPBII, and cellular retinol binding protein (CRBP)I mRNAs are found throughout the entire anterior-posterior (AP) axis of the cord, as is RA (Colbert et al. [1993] Proc. Natl. Acad. Sci. U.S.A. 90:6572-6576) and RA-sensitive transgene expression (Balkan et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:3347-3351). At E12.5, RA, transgene expression, and RAR beta become restricted to the cervical and lumbar cord. RAR alpha, CRABPI, and RXR gamma, however, are found throughout the AP extent. CRABPII and CRBPI, although expanded within the cervical and lumbar regions, are also found throughout the AP axis. Thus, several retinoid signaling molecules continue to be expressed beyond distinct regions of the spinal cord where RA is available and some RA-responsive genes are either restricted or enhanced. Exogenous RA can activate a more widespread response resulting in ectopic transgene and RAR beta expression in the thoracic and sacral cord. Not all RA-sensitive genes, however, respond; CRABPII and CRBPI expression patterns are unchanged. Finally, not every cell within the normal or exogenously induced domains of RA-dependent gene expression responds to RA, nor does every cell express RA receptors or binding proteins. Thus, regional and cellular differences in the distribution of the known retinoid receptors and binding proteins do not predict absolutely where or whether retinoid sensitive genes will be expressed or where retinoids will be available in the developing spinal cord. Instead, retinoid-mediated gene expression in the cervical and lumbar cord seems to reflect retinoid responses that rely both on the local availability of retinoids, the identity of the responding gene, and an indeterminate array of retinoid signaling molecules.

Early onset heart failure in transgenic mice with dilated cardiomyopathy.

In children, dilated cardiomyopathy is due to a variety of etiologies and usually carries a grave prognosis. The purpose of the present study was to carefully follow the progression of events leading to cardiac dilatation and congestive heart failure in a dilated cardiomyopathy model in neonatal and juvenile mice. These initial steps are often not well characterized. Furthermore, the loss of gap junctions and reduced electrical coupling of cardiomyocytes frequently found in human cardiomyopathies are also observed in these early stages. By 2 wk of age, molecular markers associated with hypertrophy were already altered. Cardiomyocyte hypertrophy, reduced connexin43 expression, and decreased conduction velocity were apparent by 4 wk, before overt cardiac dysfunction (decreased shortening fraction and chamber remodeling) that was not present until 12 wk of age. Our results show that in this model cardiomyopathic changes are present by 2 wk after birth and progress rapidly during the subsequent 2 postnatal weeks. Combined with the observations of other models of heart disease, we suggest that the first 2 wk of postnatal life are absolutely critical for normal cardiac development, and events that perturb homeostasis during this period determine whether the heart will continue to develop normally. These animals exhibit early symptoms of disease including reduced connexin43 and conduction defects before impaired cardiac function and demonstrate for the first time a temporal association between decreased connexin43 levels and the initiation of a contractility deficit that ends in heart failure.

Prothrombin deficiency results in embryonic and neonatal lethality in mice.

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII-/-) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII-/- embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII-/- embryos within this gestational time frame. However, at least one-quarter of the FII-/- mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Fatal embryonic bleeding events in mice lacking tissue factor, the cell-associated initiator of blood coagulation.

Tissue factor (TF) is the cellular receptor for coagulation factor VI/VIIa and is the membrane-bound glycoprotein that is generally viewed as the primary physiological initiator of blood coagulation. To define in greater detail the physiological role of TF in development and hemostasis, the TF gene was disrupted in mice. Mice heterozygous for the inactivated TF allele expressed approximately half the TF activity of wild-type mice but were phenotypically normal. However, homozygous TF-/- pups were never born in crosses between heterozygous mice. Analysis of mid-gestation embryos showed that TF-/- embryos die in utero between days 8.5 and 10.5. TF-/- embryos were morphologically distinct from their TF+/+ and TF+/- littermates after day 9.5 in that they were pale, edematous, and growth retarded. Histological studies showed that early organogenesis was normal. The initial failure in TF-/- embryos appeared to be hemorrhaging, leading to the leakage of embryonic red cells from both extraembryonic and embryonic vessels. These studies indicate that TF plays an indispensable role in establishing and/or maintaining vascular integrity in the developing embryo at a time when embryonic and extraembryonic vasculatures are fusing and blood circulation begins.