Three-dimensional scaffolds of fetal decellularized hearts exhibit enhanced potential to support cardiac cells in comparison to the adult.

A main challenge in cardiac tissue engineering is the limited data on microenvironmental cues that sustain survival, proliferation and functional proficiency of cardiac cells. The aim of our study was to evaluate the potential of fetal (E18) and adult myocardial extracellular matrix (ECM) to support cardiac cells. Acellular three-dimensional (3D) bioscaffolds were obtained by parallel decellularization of fetal- and adult-heart explants thereby ensuring reliable comparison. Acellular scaffolds retained main constituents of the cardiac ECM including distinctive biochemical and structural meshwork features of the native equivalents. In vitro, fetal and adult ECM-matrices supported 3D culture of heart-derived Sca-1(+) progenitors and of neonatal cardiomyocytes, which migrated toward the center of the scaffold and displayed elongated morphology and excellent viability. At the culture end-point, more Sca-1(+) cells and cardiomyocytes were found adhered and inside fetal bioscaffolds, compared to the adult. Higher repopulation yields of Sca-1(+) cells on fetal ECM relied on ß1-integrin independent mitogenic signals. Sca-1(+) cells on fetal bioscaffolds showed a gene expression profile that anticipates the synthesis of a permissive microenvironment for cardiomyogenesis. Our findings demonstrate the superior potential of the 3D fetal microenvironment to support and instruct cardiac cells. This knowledge should be integrated in the design of next-generation biomimetic materials for heart repair.

The effect of fetal anemia on fetal cardiac troponin T in pregnancies complicated by RhD alloimmunization.

OBJECTIVE: To study the effect of fetal anemia on fetal cardiac troponin T (cTnT) in pregnancies complicated by RhD alloimmunization. METHOD: Twenty pregnant women complicated by RhD alloimmunization who underwent intrauterine transfusion (IUT) for treatment of fetal anemia were studied. Immediately before IUT, fetal blood was obtained for hemoglobin and cTnT measurements. RESULTS: Complete measurements of hemoglobin and cTnT before IUT were obtained in 49 procedures, of which 20 were first-time. The regression analysis between hemoglobin z-score and cTnT values in 49 procedures showed significant negative correlation (r = -0.43, p = 0.002, Regression equation Log(cTnT) = -1.5057 + -0.07563 Hb z-score). Cardiac TnT values before first IUT were significantly associated with perinatal death. In the group with elevated cTnT (n = 7), fetal or neonatal death was more frequent (2 IUD and 2 NND) when compared to normal cTnT group (n = 13, 1 IUD) (57.1 vs. 7.7%, p = 0.031, Fisher's exact test). CONCLUSION: Fetal blood concentration of cTnT before IUT was negatively correlated to hemoglobin z-score, and levels of cTnT help to manage the pregnancies complicated by RhD alloimmunization.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome.

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development.

Normal distribution of melanocytes in the mouse heart.

We report the consistent distribution of a population of pigmented trp-1-positive cells in several important septal and valvular structures of the normal mouse (C57BL/6) heart. The pigmented cell population was first apparent by E16.5 p.c. in the right atrial wall and extended into the atrium along the interatrial septum. By E17.5, these cells were found along the apical membranous interventricular septum near or below the surface of the endocardium. The most striking distribution of dark pigmented cells was found in the tricuspid and mitral valvular leaflets and chordae tendineae. The normal distribution of pigmented cells in the valvuloseptal apparatus of C57BL/6 adult heart suggests that a premelanocytic lineage may participate in the earlier morphogenesis of the valve leaflets and chordae tendineae. The origin of the premelanocyte lineage is currently unknown. The most likely candidate populations include the neural crest and the epicardially derived cells. The only cell type in the heart previously shown to form melanocytes is the neural crest. The presence of neural crest cells, but not melanocytes, in some of the regions we describe has been reported by others. However, previous reports have not shown a contribution of melanocytes or neural crest derivatives to the atrioventricular valve leaflets or chordae tendineae in mouse hearts. If these cells are of neural crest origin, it would suggest a possibly greater contribution and persistence of neural crest cells to the valvuloseptal apparatus than has been previously understood.

Microarray gene expression profiles in dilated and hypertrophic cardiomyopathic end-stage heart failure.

Despite similar clinical endpoints, heart failure resulting from dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) appears to develop through different remodeling and molecular pathways. Current understanding of heart failure has been facilitated by microarray technology. We constructed an in-house spotted cDNA microarray using 10,272 unique clones from various cardiovascular cDNA libraries sequenced and annotated in our laboratory. RNA samples were obtained from left ventricular tissues of precardiac transplantation DCM and HCM patients and were hybridized against normal adult heart reference RNA. After filtering, differentially expressed genes were determined using novel analyzing software. We demonstrated that normalization for cDNA microarray data is slide-dependent and nonlinear. The feasibility of this model was validated by quantitative real-time reverse transcription-PCR, and the accuracy rate depended on the fold change and statistical significance level. Our results showed that 192 genes were highly expressed in both DCM and HCM (e.g., atrial natriuretic peptide, CD59, decorin, elongation factor 2, and heat shock protein 90), and 51 genes were downregulated in both conditions (e.g., elastin, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). We also identified several genes differentially expressed between DCM and HCM (e.g., alphaB-crystallin, antagonizer of myc transcriptional activity, beta-dystrobrevin, calsequestrin, lipocortin, and lumican). Microarray technology provides us with a genomic approach to explore the genetic markers and molecular mechanisms leading to heart failure.

Carp, a cardiac ankyrin-repeated protein, and its new homologue, Arpp, are differentially expressed in heart, skeletal muscle, and rhabdomyosarcomas.

Arpp, a protein containing an ankyrin repeat domain, PEST sequence, and proline-rich region, is a novel ankyrin-repeated protein highly homologous to Carp, which is proposed to be the putative genetic marker for cardiac hypertrophy. In this study, we comparatively analyzed expression of Arpp and Carp protein in skeletal and cardiac muscles and rhabdomyosarcomas (RMSs). In adult skeletal muscle, Arpp was preferentially expressed in the nucleus and cytoplasm of type I fibers, whereas Carp was barely detectable in skeletal muscle. On the other hand, in adult cardiac muscle, interestingly, Arpp was expressed in ventricles mostly, whereas Carp was expressed throughout the atrium and ventricle. Furthermore, although Carp was identified in fetal heart at 11 developmental weeks, Arpp was very low or undetectable in these fetal hearts. These results suggest that Arpp and Carp are differentially expressed and function in both skeletal and cardiac muscle of fetus and adult. We found that Arpp expression was induced during the differentiation of C2C12 cells in vitro, suggesting that Arpp-expression may be associated with the differentiation stage during myogenesis. Both Arpp and Carp were found to be expressed in all of the RMS cases studied. Because the expression patterns of Arpp in RMS were different from those of muscle actin or desmin, Arpp may be detectable in RMS cases that do not express other existing RMS markers.

DNA content and expression of cell cycle proteins in caterpillar nuclei from fetal human cardiac myocytes.

Caterpillar nuclei (CN) are characterized by their peculiar morphology, with chromatin distributed in clusters and running along the longitudinal axis of the nucleus. They can be observed in normal hearts of fetuses as well as in hearts of children and adults with rheumatic heart disease. This study has demonstrated by means of ploidy studies with digital image analysis that in the fetal heart (20.5+/-1.8 weeks) the CN (diploid = 5.6+/-8.4%; tetraploid = 46.2+/-24.2%; hypertetraploid = 46.9+/-26.3%) present higher DNA content than non-caterpillar myocyte nuclei (diploid = 89.4+/-6.2%; tetraploid = 10.0+/-4.1%; hypertetraploid = 1.5+/-1.3%) (P=0.000001, 0.00013, and 0.000038, respectively). Expression of proliferation cell nuclear antigen (PCNA; 30.6+/-11.7% in CN and 13.4+/-7.3% in non-caterpillar myocyte nuclei; P=0.0115) and cyclin B1 (2.8+/-3.8% and 12.6+/-15.6%, respectively; P=n.s.) was also positive in these nuclei. In conclusion, these results suggest that there exists a relationship between CN morphology and myocyte replication phenomena.

The effect of in utero hypoxia on fetal heart and brain trace metals.

This study determined the effect of in utero hypoxia on fetal heart and brain pro- and antioxidant trace metals. Dunkin-Hartley guinea pigs (50-60 days gestation) were exposed to 1 h hypoxia (7% O2/93% N2) followed by 4 h reoxygenation in room air. Fetal hearts and brains were harvested and analyzed for copper, iron, magnesium and zinc. Fetal brain iron was significantly increased 28% after hypoxia and 35% by 1 h posthypoxia. Fetal brain magnesium demonstrated progressive decreases of 18% by 4 h posthypoxia. No significant effects of hypoxia were observed on heart trace metals. These results indicate that prooxidant metals may be increased and antioxidant metals may be decreased in posthypoxic fetal brain during a time when these tissues may be vulnerable to oxidative injury.

Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart.

During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca(2+)) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca(V)3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca(V)3.1d is a splice variant of the alpha1G, T-type Ca(2+) channel. Immunohistochemical localization showed expression of alpha1G Ca(2+) channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the alpha1G channels. Dihydropyridine-resistant inward Ba(2+) or Ca(2+) currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the alpha1G channel expressed in human embryonic kidney-293 cells. Ni(2+) blockade discriminates among T-type Ca(2+) channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca(2+) handling proteins. We demonstrate that 100 micromol/L Ni(2+) partially blocked alpha1G currents under physiological external Ca(2+). We conclude that alpha1G T-type Ca(2+) channels are functional in midgestational fetal myocardium.

Extra-pituitary growth hormone in peripheral tissues of early chick embryos.

Early embryonic growth is independent of pituitary growth hormone (GH), since it occurs prior to the differentiation of pituitary somatotrophs. Embryogenesis is therefore thought to be regulated by local growth factors. As GH is now known to be produced in many extrapituitary sites, in which it acts in an autocrine or paracrine manner, the possibility that extra-pituitary GH may participate in embryogenesis and organogenesis was assessed by determining the immunocytochemical presence and location of GH- and GH-receptor (GHR)-like proteins in the peripheral tissues of chick embryos during their 21-day incubation period. Immunoreactive (IR)-GH, detectable by a monoclonal and two polyclonal antibodies for chicken GH, was specifically and ubiquitously present in tissues of 3-day-old embryos. At embryonic day (ED) 5, IR-GH was widespread in ectodermal, mesodermal and endodermal tissues, but it was not present in every cell of each tissue. IR-GH was particularly abundant i! n the neural tube, notochord, limb bud, somites, heart, stomach, liver, kidney, Wolffian duct and the amnion. By ED8, IR-GH was still widespread and was now present in limb bud cartilage, although the heart and liver were no longer GH immunoreactive. GH receptor immunoreactivity was also present in most tissues and cells of ED3-ED8 embryos. These results demonstrate that extrapituitary GH is abundantly present during early embryogenesis, prior to the differentiation of pituitary somatotrophs (at ED12). Since GH- and GHR-like proteins are present in most tissues of the chick embryo, it is proposed that extrapituitary GH may act as a local growth factor during embryonic development.

The effect of intermittent umbilical cord occlusion on insulin-like growth factors and their binding proteins in preterm and near-term ovine fetuses.

Intermittent umbilical cord compression with resultant fetal hypoxia can have a negative impact on fetal growth and development. Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are the most important regulators of fetal growth. In preterm (107-108 days of gestation) and near-term (128-131 days of gestation) ovine fetuses, we have determined the effect of intermittent umbilical cord occlusion (UCO) over a period of 4 days on the profile and expression of IGFs and IGFBPs. In experimental group animals (preterm n=7; near term n=7) UCOs were carried out by complete inflation of an occluder cuff (duration 90 s) every 30 min for 3-5 h each day, while control fetuses (preterm n=7; near term n=7) received no UCOs. Ewes were euthanized at the end of day 4, and fetal heart, lung, kidney, liver, skeletal muscle and placenta were collected. During UCOs, PO(2! ) fell (by approximately 13 mmHg), pH fell (by approximately 0.05) and PCO(2) increased (by approximately 7 mmHg), and changed to a similar extent in both preterm and near-term groups. In both preterm and near-term groups, there was no difference in fetal body or organ weight between UCO and control fetuses. No significant changes were observed in plasma IGF-I and -II concentrations or IGFBP-1, -2, -3 or -4 levels throughout the 4-day study at either gestational age. In the preterm group UCO fetuses, IGF-II mRNA (1.2-6.0 kb) levels were lower in fetal lung (33%, P<0.05), heart (54%, P<0.01) and skeletal muscle (29%, P<0.05), but there were no differences in IGF-I mRNA levels (7.3 kb); IGFBP-2 mRNA (1.5 kb) levels were lower in the right lobe of the liver (42%, P<0.05) and kidney (22%, P<0.01), but hig! her in the heart (72%, P<0.01), while IGFBP-4 (2.4 kb) levels were lower in skeletal muscle (21%, P<0.01). In the near-term group UCO fetuses, IGFBP-2 mRNA levels were greater in the placenta (39%, P<0.05). Thus, intermittent UCO as studied has a greater effect on the expression of genes encoding certain peptides of the fetal IGF system in selected tissues in preterm fetuses than that in near-term fetuses. Altered IGFBP-2 mRNA levels with reduced IGF-II mRNA levels in selected tissues may mediate changes in growth and/or differentiation that might become apparent if the length of the UCO study were extended.

Identification and localization of TBX5 transcription factor during human cardiac morphogenesis.

Mutations in the TBX5 transcription factor gene cause human cardiac malformation in Holt-Oram syndrome. To identify and localize TBX5 during cardiac morphogenesis, we performed immunohistochemical studies of TBX5 protein cardiac expression during human embryogenesis. Specific antibody to human TBX5 was generated in rabbits with a TBX5 synthetic peptide and affinity purification of antiserum. Anti-TBX5 was used in immunohistochemical analyses of human cardiac tissue. In embryonic and adult heart, TBX5 is expressed throughout the epicardium and in cardiomyocyte nuclei in myocardium of all four cardiac chambers. Endocardial expression of TBX5 is only present in left ventricle. Asymmetric left-sided transmyocardial gradients of TBX5 protein expression were observed in embryonic but not adult hearts. Human cardiac expression of TBX5 protein correlates with the cardiac manifestations of Holt-Oram syndrome. TBX5 transmyocardial protein gradients may contribute to normal patterning of the human heart during embryogenesis.

The distribution of stanniocalcin 1 protein in fetal mouse tissues suggests a role in bone and muscle development.

We previously isolated a mammalian gene STC1 that encodes a glycoprotein related to stanniocalcin (STC), a fish hormone that plays a major role in calcium homeostasis. However, the mammalian STC1 gene is expressed in a variety of adult tissues in contrast to fish where STC is expressed only in one unique gland, the corpuscles of Stannius. This suggested that STC1 may have wider autocrine/paracrine functions in mammals. In the present study, using immunocytochemistry, we showed that STC1 protein is localized in the developing bone and muscle of the mouse fetus. During endochondral bone formation, STC1 is found principally in prechondrocytes and prehypertrophic chondrocytes. During intramembranous bone formation STC1 is present in the mesenchyme that is about to undergo ossification. STC1 is also found in the myocardiocytes of the developing heart and at all stages of differentiation from myoblasts to myotube formation in developing skeletal muscle. The specific localization of STC1 to chondrocytes and muscle cells suggests a role for this protein in chondrogenic and myogenic differentiation.

Endothelial growth factor receptors in human fetal heart.

BACKGROUND: Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. METHODS AND RESULTS: Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. CONCLUSIONS: The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.

Connexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart.

We previously demonstrated that alpha 6 (Cx45), one of the three connexins of the mammalian myocardium, is preferentially expressed in the peripheral portion of the ventricular conduction system in rats and mice. Here we report that alpha 6 is also prominently immunolocalized in the atrioventricular node and His bundle of these species. The distribution of immunolocalized alpha 6 reveals that the node and bundle form part of an extended central conductive network circumscribing the AV and outflow junctional regions of the fetal, and less continuously, the adult heart. Of the three cardiac connexins, alpha 6 is the isoform most continuously expressed by conduction tissues, and may thus account for the recently reported viability of the alpha 5 (Cx40) knockout mouse. It is concluded that alpha 6 expression is a defining feature of the heterogenous tissues comprising the atrioventricular conduction system of the rodent heart.

A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

Identification and characterization of a new human cDNA from chromosome 21q22.3 encoding a basic nuclear protein.

Congenital heart disease (CHD) affects over 40% of Down syndrome (DS) patients. The region proposed to contain the gene(s) for DS CHD has been restricted to 21q22.2-22.3, from D21S55 to MX1. The identification and functional characterization of the genes mapping to this region is a necessary step to understand the pathogenesis of CHD in DS. In an effort to contribute to the construction of a transcriptional map of the DS CHD region we have performed direct cDNA selection using a YAC contig that maps between ETS2 and D21S15 and cDNAs synthesised from fetal heart structures. Here we describe the identification and characterization of a new gene, WRB, that maps to 21q22.3 between ACTL5 and HMG 14 and appears to be widely expressed in adult and fetal tissues. The new gene encodes a basic protein of unknown function containing a tryptophan-rich carboxyl-terminal region and a potential nuclear localization signal. Immunofluorescence analysis shows a predominant localization in the cell nucleus. The understanding of the biological function of the protein product should clarify the potential role of WRB in the pathogenesis of DS CHD.

Characterisation of subtypes of the P2X and P2Y families of ATP receptors in the foetal human heart.

ATP exerts a variety of actions within the myocardium, including the regulation of coronary vascular tone and modulation of the autonomic control of the heart. In order to characterise the ATP receptor subtypes involved in these effects, degenerate oligonucleotides were used to clone receptors of both P2X and P2Y families from the human foetal heart. About 1 ng of "Quick-Clone cDNA" from foetal human heart was subjected to amplification with two pairs of degenerate oligonucleotides designed to amplify subtypes of the P2X and P2Y receptor families by means of PCR reactions. The sequence analysis of 34 and 29 clones of the P2X and P2Y receptor families, respectively, demonstrated that P2X1, P2X3 and P2X4 subtypes are present in the human foetal heart together with P2Y6, P2Y2 and P2Y4 receptors. P2X1 and P2Y4 receptor subtypes were here characterised for the first time in the human foetal heart. The present study provides the first molecular characterisation of ATP receptors in the foetal human heart. The results show that many P2 receptor subtypes are expressed in the foetal human heart, perhaps contributing to developmental processes as well as to the activity of the foetal heart.

Glycosaminoglycan analysis in amniotic fluid and in cultured fibroblasts from normal and holoprosencephalic human embryonic organs.

Glycosaminoglycans are polysaccharides involved in epithelial-mesenchymal interaction and cell differentiation and provide a meshwork which is essential to maintain a proper intercellular milieu. The development of embryonic organs can be accompanied by alterations in the glycosaminoglycan pattern. In pregnancies with malformed fetuses, there are alterations in total glycosaminoglycans and their components (chondroitin 4-6 sulphate, dermatan sulphate, and hyaluronic acid) in amniotic fluid. We examined total glycosaminoglycans and the percentage variations of the single classes in both amniotic fluid and culture medium of fibroblasts from heart, lung, and skin obtained from five normal human fetuses and one with holoprosencephaly. In the amniotic fluid total glycosaminoglycans and their sulphate classes were increased, whereas hyaluronic acid was decreased, compared with controls. The extracellular glycosaminoglycans showed hyaluronic acid reduction in skin, while chondroitin 4-6 sulphate plus dermatan sulphate and heparan sulphate were higher in skin and heart. Our data demonstrate that variations in the glycosaminoglycan pattern are associated with alterations of the cellular environment, which can prevent normal organogenesis.

Vinculin is an essential component for normal myofibrillar arrangement in fetal mouse cardiac myocytes.

Vinculin is a cytoskeletal protein that is believed to be an essential component in the linkage of cytoskeletal actin filaments to the plasma membrane. To investigate the precise function of vinculin in the development of cardiac myofibrils, antisense oligodeoxynucleotides complementary to vinculin mRNA were used to perturb the expression of the protein during myofibril assembly and arrangement in mouse cardiac myocytes. Fetal (day 18-20 post-conception) mouse cardiac myocytes were isolated by collagenase digestion, separated by Percoll density gradient centrifugation, and plated on aligned collagen gels. By 72 h of culture, mouse myocytes displayed an elongated in vivo-like phenotype in parallel with the aligned fibrils of the collagen gels with polarized arrays of myofibrils. Two different antisense oligonucleotides (20-mer) altered the formation of the tissue-like phenotype of myocytes. These antisense oligonucleotides suppressed vinculin protein expression at 43.5+/-26.8% and 48.7+/-20.9% when compared to myocytes that were not treated. Examination of these myocytes by confocal scanning laser and transmission electron microscopy revealed a disruption of the aligned in vivo-like phenotype, assembly of thick and thin filaments, and formulation of Z-bands. Random sequence 20-mer oligonucleotides used as controls had little detectable effect on vinculin protein expression (94.2+/-14.8%), cell shape, normal alignment or assembly of myofibrils. These results indicate that vinculin is a critical cytoskeletal component, that functions in the determination of cell shape and the arrangement and organization of developing myofibrils.