N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Carbonylation of atrial myosin prolongs its interaction with actin.

Carbonylation induced by hyperthyroidism suppresses force generation of skeletal myosin and sliding velocity of actin filaments in an in vitro motility assay. However, its effects on cardiac myosin at the molecular level have not been studied. Hyperthyroidism induces a change in expression of myosin heavy chains in ventricles, which may mask the effect of oxidation. In contrast to ventricular myosin, expression of myosin heavy chains in the atrium does not change upon hyperthyroidism and enables investigation of the effect of oxidation on cardiac myosin. We studied the influence of carbonylation, a type of protein oxidation, on the motor function of atrial myosin and Ca2+ regulation of actin-myosin interaction at the level of isolated proteins and single molecules using an in vitro motility assay and an optical trap. Carbonylation of atrial myosin prolonged its attached state on actin and decreased maximal sliding velocity of thin filaments over this myosin but did not affect the calcium sensitivity of the velocity. The results indicate that carbonylation of atrial myosin induced by hyperthyroidism can be a rate-limiting factor of atrium contractility and so participates in the genesis of heart failure in hyperthyroidism.

Distinct interactions between actin and essential myosin light chain isoforms.

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.

Nkx genes are essential for maintenance of ventricular identity.

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.

Vulnerabilidad intrínseca del miocardio auricular como mecanismo de génesis de fibrilación auricular en el síndrome de Wolff-Parkinson-White / Intrinsic vulnerability of the atrial myocardium as genesis mechanism of atrial fibrillation in the Wolff-Parkinson-White syndrome

Los investigadores que describieron por primera vez el síndrome de Wolff-Parkinson-White (WPW) ya reconocieron la existencia de una asociación de ésta entidad con la fibrilación auricular (FA). Se han documentado episodios de FA hasta en un 30% de los pacientes con síndrome de WPW. Diversas modificaciones histológicas y electrofisiológicas del miocardio auricular, tales como, cambios fibrodegenerativos, aumento en la dispersión de los períodos refractarios, retardo en la conducción de los impulsos, conducción anisotrópica, e interacción con el sistema nervioso autonómico, se encuentran asociadas a la inducción, generación y persistencia de la FA. Mediante la estimulación auricular programada con extraestímulo simple durante el estudio electrofisiológico se pueden inducir varios parámetros de vulnerabilidad auricular aumentada. Por ejemplo, la actividad auricular repetitiva, la actividad auricular fragmentada, y el retardo en la conducción interauricular inducidos por un extraestímulo temprano en diástole en pacientes que poseen anormalidades electrofisiológicas del miocardio auricular, han sido utilizados como indicadores de vulnerabilidad auricular y como requisitos importantes para la génesis de la reentrada, y por ende, de la FA. Los pacientes con síndrome de WPW y FA paroxística poseen un número significativamente mayor de electrogramas auriculares anormalmente fragmentados y prolongados y una anormalidad electrofisiológica del músculo auricular significativamente mayor que los pacientes con síndrome de WPW sin FA paroxística. Estos resultados claramente demuestran que el miocardio auricular patológico y la vulnerabilidad intrínseca del miocardio auricular juegan un papel muy importante en el desarrollo de la FA en pacientes con el síndrome de WPW.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity.

AIMS: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac ß-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or ß-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rß-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. CONCLUSION: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Atrial contractile protein content and function are preserved in patients with coronary artery disease and atrial fibrillation.

OBJECTIVES: Atrial fibrillation (AF) causes atrial contractile dysfunction. The focus of this study was to determine whether the contractile deficit of human AF is the result of altered contractile protein abundance and/or function. METHODS: Atrial tissue from patients with chronic AF undergoing open-heart surgery was compared with the tissue from patients in normal sinus rhythm (NSR). Myosin isoform composition and content were determined. Intact native thin filament and cardiac myosin contractile protein performance were independently assessed in an in-vitro motility assay. RESULTS: Myosin isoform expression and total myosin content were not different between AF and NSR. Calcium-activated native thin filament function was similar between AF and NSR as measured by calcium sensitivity and maximal activation. Myosin isolated from the atria of AF and NSR groups showed similar unloaded shortening speeds and isometric force generation. CONCLUSION: Unlike human ventricular dysfunction where contractile protein function is directly affected, the contractile deficit of AF is not the result of alterations in myosin content or contractile protein function.

Tyrosine hydroxylase is expressed during early heart development and is required for cardiac chamber formation.

AIMS: Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. Whereas the neuroendocrine roles of cathecolamines postnatally are well known, the presence and function of TH in organogenesis is unclear. The aim of this study was to define the expression of TH during cardiac development and to unravel the role it may play in heart formation. METHODS AND RESULTS: We studied TH expression in chick embryos by whole mount in situ hybridization and by quantitative reverse transcription-polymerase chain reaction and analysed TH activity by high-performance liquid chromatography. We used gain- and loss-of-function models to characterize the role of TH in early cardiogenesis. We found that TH expression was enriched in the cardiac field of gastrulating chick embryos. By stage 8, TH mRNA was restricted to the splanchnic mesoderm of both endocardial tubes and was subsequently expressed predominantly in the myocardial layer of the atrial segment. Overexpression of TH led to increased atrial myosin heavy chain (AMHC1) and T-box 5 gene (Tbx5) expression in the ventricular region and induced bradyarrhythmia. Similarly, addition of l-3,4-dihydroxyphenylalanine (l-DOPA) or dopamine induced ectopic expression of cardiac transcription factors (cNkx2.5, Tbx5) and AMHC1 as well as sarcomere formation. Conversely, blockage of dopamine biosynthesis and loss of TH activity decreased AMHC1 and Tbx5 expression, whereas exposure to retinoic acid (RA) induced TH expression in parallel to that of AMHC1 and Tbx5. Concordantly, inhibition of endogenous RA synthesis decreased TH expression as well as that of AMHC1 and Tbx5. CONCLUSION: TH is expressed in a dynamic pattern during the primitive heart tube formation. TH induces cardiac differentiation in vivo and it is a key regulator of the heart patterning, conferring atriogenic identity.

Cardiac myosin isoforms from different species have unique enzymatic and mechanical properties.

The mammalian heart contains two cardiac myosin isoforms: beta-myosin heavy chain (MHC) is found predominantly in the ventricles of large mammals, and alpha-MHC is expressed in the atria. The sequence identity between these isoforms is approximately 93%, with nonidentical residues clustered in discrete, functionally important domains associated with actin binding and ATPase activity. It is well-established that rabbit alpha-cardiac myosin has a 2-fold greater unloaded shortening velocity than beta-cardiac myosin but a 2-fold lower average isometric force. Here, we test the generality of these relationships for another large mammal, the pig, as well as for a small rodent, the mouse, which expresses alpha-MHC in its ventricles throughout adulthood. Hydrophobic interaction chromatography (HIC) was used to purify myosin from mouse, rabbit, and pig hearts. The superior resolving power of HIC made it possible to prepare highly homogeneous, enzymatically active myosin from small amounts of tissue. The movement of actin filaments by myosin was measured in an in vitro motility assay. The same assay could be used to determine average isometric force by loading the actin filaments with increasing concentrations of alpha-actinin to stop filament motion. We conclude that myosin from the mouse has significantly higher velocities for both alpha and beta isoforms than myosin from rabbits and pigs, even though the 2-fold difference in velocity between isoforms is maintained. Unlike the larger mammals, however, the small rodent generates the same high isometric force for both alpha and beta isoforms. Thus, nature has adapted the function of cardiac myosin isoforms to optimize power output for hearts of a given species.

Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish.

The embryonic vertebrate heart is composed of two major chambers, a ventricle and an atrium, each of which has a characteristic size, shape and functional capacity that contributes to efficient circulation. Chamber-specific gene expression programs are likely to regulate key aspects of chamber formation. Here, we demonstrate that epigenetic factors also have a significant influence on chamber morphogenesis. Specifically, we show that an atrium-specific contractility defect has a profound impact on ventricular development. We find that the zebrafish locus weak atrium encodes an atrium-specific myosin heavy chain that is required for atrial myofibrillar organization and contraction. Despite their atrial defects, weak atrium mutants can maintain circulation through ventricular contraction. However, the weak atrium mutant ventricle becomes unusually compact, exhibiting a thickened myocardial wall, a narrow lumen and changes in myocardial gene expression. As weak atrium/atrial myosin heavy chain is expressed only in the atrium, the ventricular phenotypes in weak atrium mutants represent a secondary response to atrial dysfunction. Thus, not only is cardiac form essential for cardiac function, but there also exists a reciprocal relationship in which function can influence form. These findings are relevant to our understanding of congenital defects in cardiac chamber morphogenesis.

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

Microarray expression analysis of effects of exercise training: increase in atrial MLC-1 in rat ventricles.

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca(2+) sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal alpha-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca(2+) sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.

Analysis of the energetic state of heart cells after adenovirus-mediated expression of hALC-1.

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption.

Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression.

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.

Ventricular expression of tbx5 inhibits normal heart chamber development.

The T-box gene tbx5 is expressed in the developing heart, forelimb, eye, and liver in vertebrate embryos during critical stages of morphogenesis and patterning. In humans, mutations in the TBX5 gene have been associated with Holt-Oram syndrome, which is characterized by developmental anomalies in the heart and forelimbs. In chicken and mouse embryos, tbx5 expression is initiated at the earliest stages of heart formation throughout the heart primordia and is colocalized with other cardiac transcription factors such as nkx-2.5 and GATA4. As the heart differentiates, tbx5 expression is restricted to the posterior sinoatrial segments of the heart, consistent with the timing of atrial chamber determination. The correlation between tbx5 expression and atrial lineage determination was examined in retinoic acid (RA)-treated chicken embryos. tbx5 expression is maintained throughout the hearts of RA-treated embryos under conditions that also expand atrial-specific gene expression. The downstream effects of persistent tbx5 expression in the ventricles were examined directly in transgenic mice. Embryos that express tbx5 driven by a beta-myosin heavy chain promoter throughout the primitive heart tube were generated. Loss of ventricular-specific gene expression and retardation of ventricular chamber morphogenesis were observed in these embryos. These studies provide direct evidence for an essential role for tbx5 in early heart morphogenesis and chamber-specific gene expression.

Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart.

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.

Regulation of chamber-specific gene expression in the developing heart by Irx4.

The vertebrate heart consists of two types of chambers, the atria and the ventricles, which differ in their contractile and electrophysiological properties. Little is known of the molecular mechanisms by which these chambers are specified during embryogenesis. Here a chicken iroquois-related homeobox gene, Irx4, was identified that has a ventricle-restricted expression pattern at all stages of heart development. Irx4 protein was shown to regulate the chamber-specific expression of myosin isoforms by activating the expression of the ventricle myosin heavy chain-1 (VMHC1) and suppressing the expression of the atrial myosin heavy chain-1 (AMHC1) in the ventricles. Thus, Irx4 may play a critical role in establishing chamber-specific gene expression in the developing heart.

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken.

Two unique cDNA clones containing chicken slow myosin heavy chain (MyHC) inserts have been isolated from an expression library. Immunochemical analyses of the expressed proteins using different slow MyHC specific monoclonal antibodies were consistent with the two clones encoding slow MyHC 1 (SM1) and slow MyHC 2 (SM2) protein sequences. Northern blot analyses showed that the clones hybridized with 6-kb mRNAs that are differentially expressed in developing and adult slow muscles, further supporting the conclusion that these two clones represent SM1 and SM2 cDNAs. Sequence analyses show that both clones encode the highly conserved light meromyosin portion of the sarcomeric myosin rod and are 78-81% homologous to a mammalian slow/cardiac beta-MyHC cDNA. Hybridization using PCR generated probes specific for SM1 and SM2 sequences demonstrated that the genes encoding these two slow MyHCs colocalized to an 80-kb BssHII genomic fragment. We further show that a probe specific to a third slow MyHC gene also hybridized with the same 80-kb genomic fragment. We conclude that in the chicken genome there is a slow MyHC locus containing at least three distinct slow MyHC genes.

Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart.

A unique myosin heavy chain cDNA (AMHC1), which is expressed exclusively in the atria of the developing chicken heart, was isolated and used to study the generation of diversified cardiac myocyte cell lineages. The pattern of AMHC1 gene expression during heart formation was determined by whole-mount in situ hybridization. AMHC1 is first activated in the posterior segment of the heart when these myocytes initially differentiate (Hamburger and Hamilton stage 9+). The anterior segment of the heart at this stage does not express AMHC1 although the ventricular myosin heavy chain isoform is strongly expressed beginning at stage 8+. Throughout chicken development, AMHC1 continues to be expressed in the posterior heart tube as it develops into the diversified atria. The early activation of AMHC1 expression in the posterior cardiac myocytes suggests that the heart cells are diversified when they differentiate initially and that the anterior heart progenitors differ from the posterior heart progenitors in their myosin isoform gene expression. The expression domain of AMHC1 can be expanded anteriorly within the heart tube by treating embryos with retinoic acid as the heart primordia fuse. Embryos treated with retinoic acid prior to the initiation of fusion of the heart primordia express AMHC1 throughout the entire heart-forming region and fusion of the heart primordia is inhibited. These data indicate that retinoic acid treatment produces an expansion of the posterior (atrial) domain of the heart and suggests that diversified fates of cardiomyogenic progenitors can be altered.