RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

The molecular genetics of retinoic acid receptors: cardiovascular and limb development.

The vitamin A metabolite retinoic acid (RA) is utilized as a signalling molecule in wide variety of developmental processes, defined by defects which occur after nutritional vitamin A deficiency or after exposure to excess vitamin A. We have initiated a genetic analysis of RA function through the establishment of lines of mice which carry germline mutations in the genes which encode retinoid receptors. Defects which result from developmental RA deficiency or excess have been recovered in embryos which are deficient in various combinations of retinoid receptors. In this chapter, our current understanding of the role of RA and retinoid receptors in cardiovascular and limb development are described, as for these our level of understanding is most advanced.

Angiotensin II enhancement of hormone-stimulated cAMP formation in cultured vascular smooth muscle cells.

The mechanism by which angiotensin II (ANG II) potentiates hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation was studied in cultured rat vascular smooth muscle cells. Incubation of cells for 60 s with 100 nM ANG II produced a two- to threefold enhancement of cAMP stimulation when coupled with isoproterenol, prostaglandin I2, or adenosine. ANG II also enhanced cAMP formation when adenylyl cyclase was stimulated directly with forskolin or activated through the stimulatory guanyl nucleotide-binding protein (Gs) with cholera toxin. Forskolin stimulation was increased by only 40%, but cholera toxin-stimulated cAMP formation was doubled. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) enhanced isoproterenol-stimulated cAMP by 51%, but inhibitors of protein kinase activation had little effect on ANG II enhancement of cAMP production. However, use of PMA to cause feedback inhibition of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation blocked the effect of ANG II on agonist-stimulated cAMP formation, and the time course for this effect of PMA paralleled its inhibitory effect on Ins(1,4,5)P3 production. Furthermore, chelation of intracellular Ca2+ or treatment with calmodulin antagonists also diminished the synergism between ANG II and isoproterenol for cAMP stimulation. The results indicate that ANG II enhances cAMP formation in vascular smooth muscle cells by facilitating the interaction between activated Gs and adenylyl cyclase. In addition, the data suggest that this effect of ANG II is directly related to Ins(1,4,5)P3 stimulation and appears to involve a Ca(2+)-calmodulin-dependent mechanism.

A rapid and economical method of preparing radioiodinated cyclic nucleotide derivatives for use in radioimmunoassays.

2'-O-succinyladenosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScAMP-TME) and 2'-O-succinylguanosine 3':5'-cyclic monophosphate tyrosyl methyl ester (ScGMP-TME) were radioiodinated using chloramine T and Na125I. The resulting radiolabeled cyclic nucleotide derivatives, ScAMP-125I-TME and ScGMP-125I-TME, were subsequently purified by reverse-phase chromatography on Sep-Pak C18 cartridges (Waters Associates, Milford, MA) and tested as tracers in sensitive radioimmunoassays for cAMP and cGMP, respectively. Purified ScAMP-125I-TME and ScGMP-125I-TME functioned in the respective radioimmunoassays for up to 12 weeks when suspended in a 1:1 (v:v) mixture of n-propanol and 20 mM sodium acetate, pH 6.0. Thus, this purification method enables rapid and economical preparation of tracers for cyclic nucleotide radioimmunoassays. Furthermore, our findings suggest that reverse-phase chromatography may be applicable to the purification of other small polar molecules to which tyrosyl groups have been added for the purpose of radioiodination.

Differential effect of pertussis toxin on adenosine and muscarinic inhibition of cyclic AMP accumulation in canine ventricular myocytes.

Cyclic AMP regulation by muscarinic and adenosine receptors was investigated in isolated canine ventricular myocytes. Both the muscarinic receptor agonist, carbachol, and the adenosine receptor agonist, phenylisopropyladenosine, decreased isoproterenol-stimulated cyclic AMP accumulation in a concentration-dependent manner. Carbachol was more potent than phenylisopropyladenosine and had a greater inhibitory effect. At 10(-6) M, carbachol reduced isoproterenol-stimulated cyclic AMP by 73 +/- 5% while 10(-3) M phenylisopropyladenosine was required to decrease cyclic AMP accumulation by 54 +/- 8%. Pretreatment of myocytes with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, Gi, completely abolished the effect of phenylisopropyladenosine to reduce cyclic AMP stimulation. In comparison, pertussis toxin treatment blunted the response to carbachol and shifted the dose-effect curve to the right but did not eliminate the inhibitory action of carbachol. In toxin-treated myocytes, 10(-3) M carbachol produced a 26 +/- 6% reduction of isoproterenol-induced cyclic AMP accumulation. This pertussis toxin-insensitive action of carbachol was antagonized by atropine and pirenzepine and was prevented when either of two different phosphodiesterase inhibitors. RO-20-1724 or isobutylmethylxanthine, was included in the incubation medium. The results indicate that adenosine receptor-mediated inhibition of hormone-stimulated cyclic AMP accumulation in ventricular myocytes occurs by a single, Gi-dependent mechanism while muscarinic inhibition appears to involve both Gi-dependent and Gi-independent mechanisms. The Gi-independent mechanism may reflect enhanced phosphodiesterase activity which results from the activation of muscarinic receptors.

Downregulation of atrial markers during cardiac chamber morphogenesis is irreversible in murine embryos.

Vertebrate cardiogenesis is a complex process involving multiple, distinct tissue types which interact to form a four-chambered heart. Molecules have been identified whose expression patterns co-segregate with the maturation of the atrial and ventricular muscle cell lineages. It is not currently known what role intrinsic events versus external influences play in cardiac chamber morphogenesis. We developed novel, fluorescent-based, myocardial, cellular transplantation systems in order to study these questions in murine embryos and report the irreversible nature of chamber specification with respect to the downregulation of atrial myosin light chain 2 (MLC-2a) and alpha myosin heavy chain (alpha-MHC). Grafting ventricular cells into the atrial chamber does not result in upregulation of MLC-2a expression in ventricular cells. Additionally, wild-type ventricular muscle cells grafted into the wild-type background appropriately downregulate MLC-2a and alpha-MHC. Finally, grafting of RXRalpha gene-deficient ventricular muscle cells into the ventricular chambers of wild-type embryos does not rescue the persistent expression of MLC-2a, providing further evidence that ventricular chamber maturation is an early event. These studies provide a new approach for the mechanistic dissection of critical signaling events during cardiac chamber growth, maturation and morphogenesis in the mouse, and should find utility with other approaches of cellular transplantation in murine embryos. These experiments document the irreversible nature of the downregulation of atrial markers after the onset of cardiogenesis during ventricular chamber morphogenesis and temporally define the response of cardiac muscle cells to signals regulating chamber specification.

Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis.

Mouse embryos lacking the retinoic acid receptor gene RXR(alpha) die in midgestation from hypoplastic development of the myocardium of the ventricular chambers and consequent cardiac failure. In this study, we address the issue of whether the RXRalpha gene is required in the cardiomyocyte lineage by generating mice that harbor a ventricular restricted deficiency in RXRalpha at the earliest stages of ventricular chamber specification. We first created a conditional ('floxed') allele of RXRalpha by flanking a required exon of the gene with loxP recombination sequences. To achieve ventricular myocardium-specific gene targeting, and to avoid potential transgenic artifacts, we employed a knock-in strategy to place cre recombinase coding sequences into the myosin light chain 2v (MLC2v) genomic locus, a gene which in the heart is expressed exclusively in ventricular cardiomyocytes at the earliest stages of ventricular specification. Crossing the MLC2v-cre allele with the floxed RXRalpha gene resulted in embryos in which approximately 80% of the ventricular cardiomyocytes lacked RXRalpha function, and yet which displayed a completely normal phenotype, without evidence of the wide spectrum of congenital heart disease phenotype seen in RXRa-/- embryos, and normal adult viability. We conclude that the RXRalpha mutant phenotype is not cell autonomous for the cardiomyocyte lineage, and suggest that RXRalpha functions in a neighboring compartment of the developing heart to generate a signal that is required for ventricular cardiomyocyte development and chamber maturation.

Synergistic roles of neuregulin-1 and insulin-like growth factor-I in activation of the phosphatidylinositol 3-kinase pathway and cardiac chamber morphogenesis.

Cardiac chamber morphogenesis requires the coordinated growth of both cardiac muscle and endocardial cell lineages. Paracrine growth factors may modulate the coordinated cellular specification and differentiation during cardiac chamber morphogenesis, as suggested by the essential role of endothelial-derived growth factors, neuregulin-1, and insulin-like growth factor-I. Using the whole mouse embryo culture system for delivery of diffusible factors into the cardiac chamber, neuregulin-1 was shown to promote trabeculation of the ventricular wall. Another factor, insulin-like growth factor-I, had no apparent effect by itself. Combined treatment with neuregulin-1 and insulin-like growth factor-I strongly induced DNA synthesis of cardiomyocytes and expansion of both the ventricular compact zone and the atrioventricular cushions leading to chamber growth and maturation. In cultured cardiomyocytes, combined neuregulin-1 and insulin-like growth factor-I also had a synergistic effect to promote DNA synthesis and cellular growth, which were prevented by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Adenoviral delivery of dominant negative Rac1, which acts downstream of phosphatidylinositol 3-kinase, blocked the effect of combined neuregulin-1/insulin-like growth factor-I treatment. These studies support the concept that the interaction of neuregulin-1 and insulin-like growth factor-I pathways plays an important role in coordinating cardiac chamber morphogenesis and may occur through convergent activation of phosphatidylinositol 3-kinase.

Adenylyl cyclase isoforms and vasopressin enhancement of agonist-stimulated cAMP in vascular smooth muscle cells.

The influence of arginine vasopressin (AVP) on agonist-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in vascular smooth muscle cells (VSMC) cultured from rat thoracic aorta. Incubation of VSMC with AVP for 60 s produced a 2- to 2.5-fold enhancement of isoproterenol-induced cAMP formation. AVP also increased cAMP stimulation by the prostaglandin I2 analogue iloprost. The effect of AVP to enhance agonist-stimulated cAMP formation was completely inhibited in cells pretreated with a selective antagonist of V1 vasopressin receptors but was not affected by blockade of V2 receptors. Inhibition of protein kinase C activation failed to alter the action of AVP to potentiate cAMP stimulation, but treatment of cells with calmodulin antagonists significantly attenuated this effect of the peptide. Moreover, depletion of Ca2+ stores with thapsigargin decreased AVP enhancement of isoproterenol-stimulated cAMP by > 70%. The action of AVP to increase cAMP stimulation was also demonstrated in freshly isolated strips of rat aorta where treatment with peptide produced a twofold increase in isoproterenol-stimulated cAMP formation. RNA blot analysis indicated expression in VSMC of mRNA encoding type III adenylyl cyclase, a Ca(2+)-calmodulin-sensitive isoform of the effector. Furthermore, when detergent-solubilized membrane extract was subjected to calmodulin affinity chromatography, a peak of adenylyl cyclase activity was identified which had affinity for calmodulin matrix in the presence of Ca2+. The results indicate that AVP activates V1 receptors in VSMC to enhance agonist-stimulated cAMP formation by a Ca(2+)-calmodulin-dependent mechanism and suggest that type III adenylyl cyclase may provide a focal point in the VSMC for cross talk between constrictor and dilator pathways.

Chamber specification of atrial myosin light chain-2 expression precedes septation during murine cardiogenesis.

To study the molecular mechanisms that control patterning of the heart tube during early cardiogenesis, we have used the ventricular myosin regulatory light chain (MLC-2v), which is expressed in the ventricular segment of the primitive heart tube, as a genetic marker for ventricular specification in rodents. To assess whether the atrial isoform, MLC-2a, could also serve as a chamber-specific marker, we cloned an atrial MLC-2 cDNA (554 base pairs) which displayed homology to the human MLC-2a cDNA at both the nucleotide (87%) and amino acid (95%) levels. Northern blot, reverse transcriptase-linked polymerase chain reaction, RNase protection, and Western blot analysis revealed atrial restricted expression in the adult mouse heart, very low levels in aorta, and no detectable expression in ventricle, skeletal muscle, uterus, or liver. In situ hybridization studies during mouse embryogenesis revealed cardiac specific expression throughout days 8-16 postcoitum, with atrial restricted expression from day 12 and qualitatively greater atrial expression than ventricular from day 9. Thus, preferential pattern of expression in the atria occurs prior to septation. The MLC-2a gene was differentially regulated when compared with MLC-2v expression during embryonic stem cell cardiogenesis in vitro with MLC-2a transcript levels detectable from day 6 in suspension cultures as compared with day 9 for MLC-2v. The region-specific expression of the MLC-2a and MLC-2v genes in their respective chambers during early cardiogenesis provides genetic markers for chamber specification (atrial and ventricular) in both the in vitro and in vivo context.

Differentiation of cardiomyocytes in floating embryoid bodies is comparable to fetal cardiomyocytes.

Embryonic stem cells will cluster and differentiate into embryoid bodies, which can develop spontaneous rhythmic contractions. From these embryoid bodies, cardiomyocytes can be isolated based on density by a discontinuous Percoll gradient. These cardiomyocytes differentiate into ventricular myocytes, which is demonstrated by the expression of the ventricular specific isoform of the myosin light chain 2 gene. In this study the functional expression of ion channels was compared between fetal cardiomyocytes (in vivo) and stem cell derived cardiomyocytes (in vitro). Sodium and calcium currents together with transient potassium currents could be detected in early developmental stages (

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos.

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.

Atrial-like phenotype is associated with embryonic ventricular failure in retinoid X receptor alpha -/- mice.

We have recently characterized a cardiac model of ventricular chamber defects in retinoid X receptor alpha (RXR alpha) homozygous mutant (-/-) gene-targeted mice. These mice display generalized edema, ventricular chamber hypoplasia, and muscular septal defects, and they die at embryonic day 15. To substantiate our hypothesis that the embryos are dying of cardiac pump failure, we have used digital bright-field and fluorescent video microscopy and in vivo microinjection of fluorescein-labeled albumin to analyze cardiac function. The affected embryos showed depressed ventricular function (average left ventricular area ejection fraction, 14%), ventricular septal defects, and various degrees of atrioventricular block not seen in the RXR alpha wild-type (+/+) and heterozygous (+/-) littermates (average left ventricular area ejection fraction, 50%). The molecular mechanisms involved in these ventricular defects were studied by evaluating expression of cardiac-specific genes known to be developmentally regulated. By in situ hybridization, aberrant, persistent expression of the atrial isoform of myosin light chain 2 was identified in the ventricles. We hypothesize that retinoic acid provides a critical signal mediated through the RXR alpha pathway that is required to allow progression of development of the ventricular region of the heart from its early atrial-like form to the thick-walled adult ventricle. The conduction system disturbances found in the RXR alpha -/- embryos may reflect a requirement of the developing conduction system for the RXR alpha signaling pathway, or it may be secondary to the failure of septal development.

Developmental changes in beta-adrenergic modulation of L-type Ca2+ channels in embryonic mouse heart.

In the adult mammalian myocardium, cellular Ca2+ entry is regulated by the sympathetic nervous system. L-type Ca2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca2+ entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca2+ channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch-clamp procedures to study modulation of L- type Ca2+ channel currents and to assay functional activity of distinct steps in the beta-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that the L-type Ca2+ channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca2+ channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the beta-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca2+ channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca2+ channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca2+ channel insensitivity to beta-A agonists in early stages of the developing murine heart.

Selective requirement of myosin light chain 2v in embryonic heart function.

Two major myosin light chain 2 isoforms are coexpressed in the early stages of murine cardiogenesis, a cardiac ventricular isoform and a cardiac atrial isoform, each of which is tightly regulated in a muscle cell-type-specific manner during embryogenesis (Chien, K. R., Zhu, H., Knowlton, K. U., Miller-Hance, W., van Bilsen, M., O'Brien, T. X., and Evans, S. M. (1993) Annu. Rev. Physiol. 55, 77-95). We have disrupted myosin light chain 2v gene in mice and monitored in vivo cardiac function in living myosin light chain 2v -/- embryos. The mutant embryos die at approximately embryonic day 12.5. In mutant ventricles, the myosin light chain 2a protein level is increased and reaches levels comparable to the myosin light chain 2v in the ventricles of wild type littermates and is appropriately incorporated into the thick filaments of mutant embryonic hearts. However, despite the substitution of myosin light chain 2a, ultrastructural analysis revealed defects in sarcomeric assembly and an embryonic form of dilated cardiomyopathy characterized by a significantly reduced left ventricular ejection fraction in mutant embryos compared with wild type littermates. We conclude that myosin light chain 2v may have a unique function in the maintenance of cardiac contractility and ventricular chamber morphogenesis during mammalian cardiogenesis and that a chamber-specific combinatorial code for sarcomeric assembly may exist that ultimately requires myosin light chain 2v in ventricular muscle cells.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.