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The outermost layer of the heart, the epicardium, is an essential cell population that contributes, through epithelial-to-mesenchymal transition (EMT), to the formation of different cell types and provides paracrine signals to the developing heart. Despite its quiescent state during adulthood, the adult epicardium reactivates and recapitulates many aspects of embryonic cardiogenesis in response to cardiac injury, thereby supporting cardiac tissue remodeling. Thus, the epicardium has been considered a crucial source of cell progenitors that offers an important contribution to cardiac development and injured hearts. Although several studies have provided evidence regarding cell fate determination in the epicardium, to date, it is unclear whether epicardium-derived cells (EPDCs) come from specific, and predetermined, epicardial cell subpopulations or if they are derived from a common progenitor. In recent years, different approaches have been used to study cell heterogeneity within the epicardial layer using different experimental models. However, the data generated are still insufficient with respect to revealing the complexity of this epithelial layer. In this review, we summarize the previous works documenting the cellular composition, molecular signatures, and diversity within the developing and adult epicardium., Competing Interests: The authors declare no conflicts of interest.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras, and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

microRNAs ( miRNAs ) are small non-coding RNAs required for the post-transcriptional control of gene expression. MicroRNAs play a critical role in modulating muscle regeneration and stem cell behavior. Muscle regeneration is affected in muscular dystrophies, and a critical point for the development of effective strategies for treating muscle disorders is optimizing approaches to target muscle stem cells in order to increase the ability to regenerate lost tissue. Within this framework, miRNAs are emerging as implicated in muscle stem cell response in neuromuscular disorders and new methodologies to regulate the expression of key microRNAs are coming up. In this review, we summarize recent advances highlighting the potential of miRNAs to be used in conjunction with gene replacement therapies, in order to improve muscle regeneration in the context of Duchenne Muscular Dystrophy (DMD).

Cardiovascular development is governed by a complex interplay between inducting signals such as Bmps and Fgfs leading to activation of cardiac specific transcription factors such as Nkx2.5, Mef2c and Srf that orchestrate the initial steps of cardiogenesis. Over the last decade we have witnessed the discovery of novel layers of gene regulation, i.e. post-transcriptional regulation exerted by non-coding RNAs. The function role of small non coding RNAs has been widely demonstrated, e.g. miR-1 knockout display several cardiovascular abnormalities during embryogenesis. More recently long non-coding RNAs have been also reported to modulate gene expression and function in the developing heart, as exemplified by the embryonic lethal phenotypes of Fendrr and Braveheart knock out mice, respectively. In this study, we investigated the differential expression profile during cardiogenesis of previously reported lncRNAs in heart development. Our data revealed that Braveheart, Fendrr, Carmen display a preferential adult expression while Miat, Alien, H19 preferentially display chamber-specific expression at embryonic stages. We also demonstrated that these lncRNAs are differentially regulated by Nkx2.5, Srf and Mef2c, Pitx2 > Wnt > miRNA signaling pathway and angiotensin II and thyroid hormone administration. Importantly isoform-specific expression and distinct nuclear vs cytoplasmic localization of Braveheart, Carmen and Fendrr during chamber morphogenesis is observed, suggesting distinct functional roles of these lncRNAs in atrial and ventricular chambers. Furthermore, we demonstrate by in situ hybridization a dynamic epicardial, myocardial and endocardial expression of H19 during cardiac development. Overall our data support novel roles of these lncRNAs in different temporal and tissue-restricted fashion during cardiogenesis., (Copyright © 2019. Published by Elsevier B.V.)

Atrial fibrillation (AF) is the most frequent arrhythmogenic disease in humans, ranging from 2% in the general population and rising up to 10-12% in 80+ years. Genetic analyses of AF familiar cases have identified a series of point mutations in distinct ion channels, supporting a causative link. However, these genetic defects only explain a minority of AF patients. Genomewide association studies identified single nucleotide polymorphisms (SNPs), close to PITX2 on 4q25 chromosome, that are highly associated to AF. Subsequent GWAS studies have identified several new loci, involving additional transcription and growth factors. Furthermore, these risk 4q25 SNPs serve as surrogate biomarkers to identify AF recurrence in distinct surgical and pharmacological interventions. Experimental studies have demonstrated an intricate signalling pathway supporting a key role of the homeobox transcription factor PITX2 as a transcriptional regulator. Furthermore, cardiovascular risk factors such as hyperthyroidism, hypertension and redox homeostasis have been identified to modulate PITX2 driven gene regulatory networks. We provide herein a state-of-the-art review of the genetic bases of atrial fibrillation, our current understanding of the genetic regulatory networks involved in AF and its plausible usage for searching novel therapeutic targets., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial-mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf-beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp- and Tgf-beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages., Results: qRT-PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR-23b, miR-199a, and miR-15a displayed increased expression during early AVC development whereas others such as miR-130a and miR-200a display decreased expression levels. Functional analyses of miR-23b, miR-199a, and miR-15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT-related genes that are predicted to be targeted by these microRNAs., Conclusions: Our data demonstrate that miR-23b and miR-199a over-expression can impair atrioventricular EMT., (© 2015 Wiley Periodicals, Inc.)

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5(+) satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aims: Ion channel remodelling and ventricular conduction system (VCS) alterations play relevant roles in the generation of cardiac arrhythmias, but the interaction between ion channel remodelling and cardiac conduction system dysfunctions in an arrhythmogenic context remain unexplored., Methods and Results: We have used a transgenic mouse line previously characterized as an animal model of Long QT Syndrome (LQTS) to analyse ion channel remodelling and VCS configuration. Reverse transcriptase-PCR and immunohistochemistry analysis showed early cardiac sodium channel upregulation at embryonic stages prior to the onset of Kv potassium channel remodelling, and cardiac hypertrophy at foetal stages. In line with these findings, patch-clamp assays demonstrated changes in sodium current density and a slowing of recovery from inactivation. Functional analysis by optical mapping revealed an immature ventricular activation pattern as well as an increase in the total left ventricle activation time in foetal transgenic hearts. Morphological analysis of LQTS transgenic mice in a Cx40(GFP/+)background demonstrated VCS dysmorphogenesis during heart development., Conclusions: Our data demonstrate early sodium channel remodelling secondary to IKs blockage in a mouse model of LQTS leading to morphological and functional anomalies in the developing VCS and cardiac hypertrophy. These results provide new insights into the mechanisms underlying foetal and neonatal cardiac electrophysiological disorders, which might help understand how molecular, functional, and morphological alterations are linked to clinical pathologies such as cardiac congenital anomalies, arrhythmias, and perinatal sudden death.

Pitx2 is a homeobox transcription factor involved in left-right signaling during embryogenesis. Disruption of left-right signaling in mice within its core nodal/lefty cascade, results in impaired expression of the last effector of the left-right cascade, Pitx2, leading in many cases to absence or bilateral expression of Pitx2 in lateral plate mesoderm (LPM). Loss of Pitx2 expression in LPM results in severe cardiac malformations, including right cardiac isomerism. Pitx2 is firstly expressed asymmetrically in the left but not right LPM, before the cardiac crescent forms, and subsequently, as the heart develops, becomes confined to the left side of the linear heart tube. Expression of Pitx2 is remodeled during cardiac looping, becoming localized to the ventral portion of the developing ventricular chambers, while maintaining a distinct left-sided atrial expression. The importance of Pitx2 during cardiogenesis has been illustrated by the complex and robust cardiac defects observed on systemic deletion of Pitx2 in mice. Lack of Pitx2 expression leads to embryonic lethality at mid-term, and Pitx2-deficient embryos display isomeric hearts with incomplete closure of the body wall. However, whereas the pivotal role of Pitx2 during cardiogenesis is well sustained, its putative role in the fetal and adult heart is largely unexplored. Recent genome-wide association studies have identified several genetic variants highly associated with atrial fibrillation (AF). Among them are genetic variants located on chromosome 4q25 adjacent to PITX2. Since then several transgenic approaches have provided evidences of the role of the homeobox transcription factor PITX2 and atrial arrhythmias. Here, we review new insights into the cellular and molecular links between PITX2 and AF.

Pitx2 is a paired-related homeobox gene that is expressed in muscle progenitors during myogenesis. We have previously demonstrated that overexpression of Pitx2c isoform in myoblasts maintained these cells with a high proliferative capacity and completely blocked terminal differentiation by inducing high Pax3 expression levels (Martinez et al., 2006). We now report that Pitx2c-mediated proliferation vs. differentiation effect is maintained during in vivo myogenesis. In vivo Pitx2c loss of function leads to a decrease in Pax3+/Pax7- cell population in the embryo accompanied by an increase of Pax3+/Pax7+ cells. Pitx2c transient-transfection experiments further supported the notion that Pitx2c can modulate Pax3/Pax7 expression. Pitx2c but not Pitx3 controls Pax3/Pax7 expression, although redundant roles are elicited at the terminal myoblast differentiation. Contrary to Pitx2c, Pitx3 does not regulate cell proliferation or Pax3 expression, demonstrating the specificity of Pitx2c mediating these actions in myoblasts. Furthermore we demonstrated that Pitx2c modulates Pax3 by repressing miR27 expression and that Pax3-miR-27 modulation mediated by Pitx2c is independent of Pitx2c effects on cell proliferation. Therefore, this study sheds light on previously unknown function of Pitx2c balancing the different myogenic progenitor populations during myogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aim: The knowledge of the molecular signals that control cell differentiation into cardiomyocytes is critical to apply cell-based therapies and repair an injured heart. The transcription factor Pitx2 has essential roles in the development of different organs including the heart. Although a direct role of Pitx2 in the developing myocardium has recently been reported, the molecular pathways driven by Pitx2 as well as its cardiac target genes remain largely unexplored. The aim of this study was to unravel the molecular mechanisms driven by Pitx2 during the process of cardiomyocyte differentiation in vitro in mouse embryonic stem cell-derived cardiomyocytes., Methods and Results: Pitx2c was overexpressed in the R1-embryonic stem cell line. mRNA levels and protein distribution of several specific cardiac genes were analyzed by real-time PCR and immunohistochemistry experiments in R1-embryonic stem cell-derived beating areas at different stages of in vitro differentiation. Our results show that overexpression of Pitx2c in embryonic stem cell-derived cardiomyocytes is able to dynamically upregulate several cardiac-enriched transcription factors such as Isl1, Mef2c and Gata4. Additionally, Pitx2c induces the expression of chamber-specific cardiac genes such as Tbx5, Nppa and Cx40. These data were validated in an in vivo model of Pitx2 loss of function., Conclusion: Taken together, these results demonstrate that Pitx2 plays a major role reinforcing the transcriptional program of cardiac differentiation., (Copyright © 2011 S. Karger AG, Basel.)

The detection and analysis of protein isoforms is a complicated task especially if they differ only in small specific domains. Obtaining specific polyclonal antibodies against these domains is a challenge, but if successful it can have a wide range of applications, such as in proteomics and immunochemical analysis. We show herein a method of overexpression and purification of two small specific domains corresponding to the isoforms b and c of the murine transcription factor Pitx2, and the generation and purification of monospecific polyclonal antibodies against them, by using a two-step affinity purification procedure, based on the use of CNBr-Sepharose matrix. Such a method also allows recovering monospecific polyclonal antibodies against the tag fusion peptide (C-LYTAG tag). The specificity of the isolated polyclonal antibodies was demonstrated by Western blot and immunohistochemical analysis. In addition, our protocol is easily scalable and allows the generation of monospecific polyclonal antibodies for large-scale analysis.

Cardiac differentiation involves cross-regulation of several transcription factors, such as Mef2C, regulated by p38alpha MAP kinase. We analysed the role of p38alpha in cardiac differentiation. Either the absence or inhibition of p38alpha impairs MEF2C nuclear localization in cardiomyocytes, colocalising with vimentin at the perinuclear region. As a consequence, expression of the Mef2C targets, ANF and myocardin, is drastically downregulated. In contrast, Mlc2v and crt are mainly unaltered, probably by the strong Mef2B upregulation, conpensating for the impaired Mef2C transactivity. In addition, p38alpha deficiency leads to a decrease in the phosphorylated Mlc2v fraction and alpha-actinin accumulation causing sarcomere disorganisation. We propose a critical role for p38alpha in early stages of cardiac differentiation by modulation of Mef2C localisation and sarcomeric assembly.

Aims: The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis., Methods and Results: Scn5a mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs., Conclusion: Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line., ((c) 2006 Wiley-Liss, Inc.)

Objectives: The aim of this study is to analyze Scn1b mRNA expression levels and protein distribution of Scn1b, a putative modulator of the pore-forming Na(+) channel subunit in the heart, during mouse cardiac development., Methods: Scn1b mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E18.5 as well as in postnatal and adult heart. Scn1b protein distribution and subcellular localization during cardiogenesis were analyzed by immunohistochemistry and confocal microscopy., Results: Scn1b mRNA showed a dynamic expression pattern, peaking at stage E12.5 and decreasing at E15.5. Scn1b mRNA increased at later embryonic and neonatal stages, being maximal in the adult heart. Immunohistochemistry experiments revealed comparable distribution of Scn1b protein between the different cardiac chambers at early embryonic stages. With further development, Scn1b protein showed an enhanced expression in the trabeculated myocardium and the bundle branches. At the subcellular level in later embryonic and postnatal mouse cardiomyocytes, Scn1b was present in T-tubules as identified by immunostaining of alpha-actinin, and in the intercalated disks as identified by immunostaining of connexin 43., Conclusion: These results demonstrate that Scn1b is expressed during mouse heart development, suggesting it can play an important role in the action potential configuration of the cardiomyocytes during heart morphogenesis.

Retinoic acid (RA), the active metabolite of vitamin A, plays a significant role in regulating cardiac form and function throughout the life of the organism. Both cardiac morphogenesis and myocardial differentiation are affected by alterations in RA homeostasis. In order to test the effect of all-trans RA and 13-cis RA on cardiomyocyte differentiation, we studied the level and the subcellular compartmentalization of alpha-tropomyosin and troponin-T proteins in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the levels of alpha-tropomyosin and troponin-T in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The greatest increases in alpha-tropomyosin occurred in the cytoplasmic fraction in HH22 cells cultured for 24 h with all-trans RA or 13-cis RA, whereas the greatest increases in troponin-T were found in the cytoplasmic fraction of HH32 cells exposed to retinoids for 24 h. In cultures treated for 48 h with retinoids, the levels of alpha-tropomyosin and troponin-T showed significant increases in the cytoplasmic compartment of cells treated in HH32-with respect to the control values. These findings are further evidence that RA plays a modulating role in the formation and reorganization of sarcomeric proteins during the process of cardiomyocyte maturation., (Copyright 2003 S. Karger AG, Basel)

The in vitro study of mechanisms involved in drug-induced maturation has made it possible to use differentiation-based therapy in clinical practice. The goal of this new therapy is the development of specific agents to induce cancer cells to stop proliferating and express characteristics of normal cells. Recently, by structural modifications of 5-fluorouracil (5-FU), we synthesized a new pyrimidine acyclonucleoside-like compound, 1-¿[3-(3-chloro-2-hydroxypropoxy)-1-methoxy]propyl¿-5-fluorouracil (QF-3602), which showed in rhabdomyosarcoma cells a low toxicity and time-dependent growth inhibition. In this work, we compared the degree of myogenic differentiation of RD rhabdomyosarcoma (RMS) cells after treatment with QF-3602 and 5-FU. Scanning and transmission electron microscopy (SEM and TEM) and immunocytochemical analyses showed that QF-3602 induced the appearance of myofilaments along the myotube-like giant RD cells, an increase in fibronectin and a decrease in vimentin expression. In contrast, only minor changes were observed with 5-FU. Moreover, polymerase chain reaction (PCR) analyses showed that QF-3602 did not induce overexpression of the mdr 1 gene, a resistance mechanism that frequently appears in classical cytotoxic therapy in these tumors. Compounds obtained by structural modifications of 5-FU may be useful in differentiation therapy as a new approach to the treatment of RMS.

Recent studies suggest that peptide growth factors play a functional role in cardiac muscle. To test whether embryonic cardiac muscle is a target for regulation by basic fibroblast growth factor and platelet-derived growth factor, we analyzed the effects of these peptides on the expression of the intermediate filaments desmin and vimentin at the subcellular level during development. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting and fluorescence-activated cell sorting analysis were used to study the effect of basic fibroblast growth factor and platelet-derived growth factor on cultures of chick cardiomyocytes during development. Cytoplasmic and cytoskeletal concentrations of desmin and vimentin were dependent on the stage of embryonic development and on the type of growth factor added to the culture. The most significant finding was the increase in desmin expression in the cytoplasmic and cytoskeletal compartments after treatment with basic fibroblast growth factor (10 ng/ml) of chick heart cells at Hamburger and Hamilton stage 19. In more mature stages, basic fibroblast growth factor did not modify the levels of desmin expression. However, this factor led to a progressive deceleration in the rate of increase in vimentin expression. Platelet-derived growth factor increased vimentin expression in all stages studied, the greatest increases appearing in early stages of heart development. Our findings support the hypothesis that basic fibroblast growth factor plays a role in cardiomyocyte differentiation during the early stages of development, whereas platelet-derived growth factor has a dedifferentiating effect., (Copyright 2000 S. Karger AG, Basel)

Early heart development is known to be sensitive to retinoid concentrations. Although the influence of retinoids on cardiac morphogenesis has been described previously, the effect of retinoids on cardiomyocyte differentiation during development has not been characterized. We quantified the effects of the retinoic acids all-trans RA and 13-cis RA on alpha-actin and alpha-actinin at the subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the concentration of alpha-actin and alpha-actinin in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The effect was greatest in cardiomyocytes treated for 24 h with all-trans RA and in cells from HH22 embryos. The greatest increases in alpha-actin concentration occurred in the cytoskeletal fraction of HH22 cells cultured for 24 h with all-trans or 13-cis RA, whereas the greatest increases in alpha-actinin were found in the cytoplasmic fraction of HH22 cells exposed to retinoids for 24 h. We conclude that retinoic acid plays a role in the reorganization of the pattern of sarcomeric protein expression during cardiomyocyte differentiation.

We used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of myocardial proteins followed by Western blotting to study the formation of antiheart antibodies during three months after myocardial infarction and the relationship between the appearance of antibodies and clinical and laboratory findings. Fifty-four percent of the 66 patients with infarction had different types of antiheart antibodies. The autoantibodies detected most frequently were against 35 and 42 kDa cardiac proteins. Immunoblottings with purified proteins showed that these autoantibodies reacted against myocardial tropomyosin and actin, which have been detected after acute myocardial infarction and can have immunogenetic activity through a humoral immune response. However, only the presence of autoantibody against myocardial tropomyosin correlated significantly with the presence of clinical and laboratory findings. Our results suggest that autoantibody against myocardial tropomyosin may play an immunopathogenic role in the development of symptoms in these patients.

This study analyses the NORs of Tapinoma nigerrimum, a species that, as known from previous studies, has various chromosomes which carry a NOR site. The analysis was made by a combination of three methods: silver nitrate staining, in situ hybridization with fluorescein-or digoxigenin-labelled probes, and staining with the CG-specific fluorochrome chromomycin A3. The silver staining technique showed an Ag-positive region on chromosome 6 and on various other chromosomes. However, the application of in situ hybridization techniques showed only one positive signal in the proximal region of the short arm of chromosome 6 of T. nigerrimum. Similar results were observed by CMA banding. The absence of rDNA genes or the presence of only a small number of these, not detectable with the above probes, might explain the absence of hybridization signal in the remaining chromosomes.

Gene transfection has been accomplished with a variety of techniques such as DEAE dextran, calcium phosphate coprecipitation, protoplast fusion, liposomes, microinjection and recombinant bacteriophages. However, transfection by electroporation, consisting of the reversible permeabilization of cell membranes after exposure to a pulsed electric field, has been shown to be the most rapid, simple and efficient method for the stable incorporation of genes in different cell lines. We studied rhabdomyosarcoma cells subjected to electroporation in two different vol. [400 microliters (group 1) and 150 microliters (group 2] of 140 mM NaCl/15 mM Hepes buffer, pH 7.2) and evaluated the effects of electroporation volume on growth and differentiation. Low sample volumes induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death. Our results suggest that in electroporation low sample vol. of rhabdomyosarcoma cells induced morphological and phenotypic differentiation, with increased expression of desmin, alpha-actinin and tropomyosin.

Cytotoxic agents used in cancer therapy may induce differentiation in tumour cells with no proliferative potential. However, chemotherapy can also induce multidrug resistance, a formidable obstacle to the successful treatment of tumours. Both events were recently shown to occur in a rhabdomyosarcoma cell line (RD-DAC) resistant to actinomycin D, a drug of choice in the treatment of these tumours. To analyse this connection, cell line RD cultures were investigated with progressive concentrations of actinomycin D and it was shown that a minimum dose (1.2 x 10(-6) mM) of the drug was necessary to increase mdr 1 mRNA in RD-DAC. The mechanism of mdr 1 overexpression was an increase in the number of copies of the mdr 1 gene, although the mRNA levels were not correlated with mdr 1 amplification. Drug resistance mediated by mdr 1 overexpression coincided with the development of myogenic differentiation in RD-DAC and with a decrease in c-myc mRNA levels, whereas levels of N-myc mRNA showed no modulation. These findings suggest that factors implicated in cell proliferation and differentiation, such as c-myc, may be responsible for the control of genes related to the development of multidrug resistance in rhabdomyosarcomas. Modulation of these factors may determine the sensitivity of rhabdomyosarcoma cells to drugs and may play an important role in triggering the differentiation programme found in these resistant rhabdomyosarcoma cells.

Neoplastic transformation may be an alteration in the process of cell maturation that leads to an infinite capacity for proliferation. Because the cytodestruction caused by most drugs available for cancer chemotherapy is often accompanied by significant morbidity and poor response, the induction of differentiation has been proposed as an alternative approach to conventional anticancer therapy. We used human rhabdomyosarcoma cell line RD to analyze the differentiation process induced by actinomycin D, a drug of choice in the conventional treatment of rhabdomyosarcomas. Low concentrations of actinomycin D induced a terminal process of morphological and ultrastructural myogenic differentiation in rhabdomyosarcoma cells, which concluded with cell death. However, this potential therapeutic effect cannot be considered complete because of the presence of tumoral cells that are heterogeneous with respect to actinomycin D chemosensitivity. This heterogeneity led to the appearance of foci of resistant cells which, despite their greater degree of differentiation in comparison with the parental cell line, escaped from terminal myogenic differentiation. This subgroup of tumoral cells may be responsible for the failure of cytotoxic treatment.

To test whether cardiac muscle is a target for regulation by peptide growth factors, we analyzed the effects of two growth factors on actin and alpha-actinin expression at the subcellular level. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting, and fluorescense-activated cell sorter analysis were used to quantify the effects of basic fibroblast growth factor and platelet-derived growth factor on cultures of chick myocardiocytes during development. Cytoplasmic and cytoskeletal concentrations of actin and alpha-actinin were dependent on the stage of embryonic development and on the type of growth factor added to the culture. The most significant finding was the increase in actin and alpha-actinin expression in the cytoplasmic compartment after treatment with basic fibroblast growth factor of chick heart cells at Hamburger and Hamilton's stage 19. At stage 39, basic fibroblast growth factor induced less marked changes in the accumulation of actin and alpha-actinin. Platelet-derived growth factor decreased alpha-actinin expression slightly in the cytoskeletal compartment in more mature stages of heart development. Our findings support the hypothesis that basic fibroblast growth factor plays a role in cardiomyocyte differentiation during the early stages of development.

The right ventricle was studied in 75 anatomically normal swine hearts, using, in all, nine geometric and volumetric parameters: ventricular-wall thickness, length of the right-ventricular inflow and outflow tracts, and volume of the right-ventricular inflow and outflow tracts. The data for these parameters were compared with previously published patterns for human hearts and volumetric data were compared with patterns of normality found in human hearts. As in the human heart, the ventricular inflow tract in swine hearts was significantly shorter than the outflow tract (P < 0.0001).

In the present study we quantified the contractile protein troponin-T at the cellular and subcellular level in chick embryo cardiomyocytes to investigate the modulation of cardiac development by catecholamines. We analyzed the effects of these drugs on cultures of chick cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 21, HH stage 29 and HH stage 40 embryos; cardiomyocytes are considered to be mature at HH stage 40. We analyzed the modifications these drugs induced in the transcription of the gene for chick cardiac troponin-T. Sodium dodecyl sulfate-gel electrophoresis and immunobloting showed that cytoplasmic and cytoskeletal concentrations of troponin-T are dependent on the stage of embryonic development analyzed, and on the type of catecholamine added to the culture. The most significant finding was the increase in troponin-T mRNA in the chick heart at HH stage 40, accompanied by an increase in the increase in the expression of this protein in the cytoskeletal compartment after treatment with norepinephrine. At HH stage 21, norepinephrine induced less marked changes in the accumulation of troponin-T in comparison with untreated cardiomyocytes.

We quantified the effect of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) on the contractile protein troponin-T (TnT) at the cellular and subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 19, 29, and 39 embryos. Because expression of thin-filament molecules is considered a good marker of differentiation in muscle cell cultures, we analyzed the modifications these growth factors induced in the transcription of the gene for chick cardiac TnT. Sodium dodecyl sulfate-polyacrylamate gel electrophoresis (SDS-PAGE) and immunoblotting showed that cytoplasmic and cytoskeletal concentrations of TnT are dependent on the stage of embryonic development analyzed and on the type of growth factors added to the culture. The most significant finding was the increase in TnT expression in the cytoplasmic compartment (p < 0.001), accompanied by a slight increase in TnT mRNA, after treatment with bFGF of chick heart cells obtained at HH stage 19. At HH stage 39, bFGF induced less marked changes in the accumulation of TnT in comparison with untreated cardiomyocytes. Our findings support the hypothesis that bFGF plays a role in cardiomyocyte differentiation during early stages of development.

We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and diacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.

The right ventricle was studied in 75 anatomically normal swine hearts. Nine parameters in the papillo-tendino valvular system and three corresponding to the tricuspid orifice, pulmonary orifice and length of the inflow tract were measured. Correlations were established between the parameters and heart weight in grams, between the different parameters themselves, and between heart weight and body weight. The results were compared with similar data from human hearts, and were considered of use to researchers planning to use the swine heart as an experimental model to study congenital or induced heart diseases, or as a reference for the clinical interpretation of spontaneous cardiac, anomalies in swine.

We used Western blot, a highly sensitive technique that detects amounts of protein as low as 0.1 to 1.0 ng, to investigate the possible presence in the blood stream of the contractile protein alpha-actin in 29 patients diagnosed with angina pectoris (Braunwald's classification). Circulating protein was identified with a monoclonal antibody specific for cardiac alpha-actin. Of the 20 control samples of blood, the immunoblot results were negative for alpha-actin in 19. Of the 30 patients with skeletal muscle damage caused by surgery, 27 were negative for circulating alpha-actin. Of the 29 patients with angina pectoris, circulating alpha-actin was found in 19 as a 43 kDa band in immunoblots. Of the four patients with anterior acute myocardial infarction, mean concentration of circulating alpha-actin was 58 mg/l. Among the patients with angina pectoris, the highest circulating concentrations (mean 40 mg/l) was found in those with prolonged angina (class III B, according to Braunwald's classification). In the entire group of individuals with angina pectoris alpha-actin was detectable in serum for up to 175 h after the onset of pain, and showed two peaks, one at 1 h (112 mg/l) and one at 50 h (82 mg/l) after the onset of pain. These findings reinforce the notion that unstable angina should be considered a serious condition.

We analyzed the influence of 6 and 24 h of treatment with the fibric acid derivatives bezafibrate (10 micrograms/ml), gemfibrozil (23 micrograms/ml), and fenofibrate (30 micrograms/ml) on alpha-actinin, troponin-T, and tropomyosin proteins in the cytoplasmic and cytoskeletal fractions of cultured chick myocardiocytes. The findings with sodium dodecyl sulfate-gel electrophoresis and immunoblotting showed that all three drugs modified cellular and subcellular protein levels in different ways: bezafibrate and fenofibrate produced the most significant alterations in both fractions, modifying alpha-actinin, troponin T, and tropomyosin compartmentalization in myocardiocytes, whereas gemfibrozil altered these proteins less notably. Given the role of these proteins in heart muscle contraction, fibric acid derivative-induced changes may be related with the secondary effects of these drugs on heart rhythmicity.

We used Western-blot analysis to investigate the possible presence in the bloodstream of the contractile protein alpha-actin in 70 patients diagnosed with acute myocardial infarction on the basis of clinical, electrocardiographic and laboratory (creatine kinase and lactate dehydrogenase) criteria. Circulating protein was identified with a monoclonal antibody specific for cardiac alpha-actin. Of the 70 control samples of blood, the immunoblot results were negative for alpha-actin in 98% of the cases. Of the 30 patients with skeletal muscle damage caused by surgery, 26 were negative for circulating alpha-actin. Of the 70 patients with acute myocardial infarction, circulating alpha-actin was found in 67 (95%) as a 43 kDa band in immunoblots; the highest circulating concentrations (0.0580 micrograms/microliters) were found in those with anterior acute myocardial infarction. Circulating alpha-actin was detected in samples taken between 1 and 180 h after the onset of pain, and showed a biphasic pattern of appearance. Our findings for serum alpha-actin, together with the relationship between serum concentrations of this protein and sex (p = 0.001), tobacco use (p = 0.007) and postepisode complications (p = 0.002), should make it possible to gain a deeper understanding of acute myocardial infarction as a clinical entity.

We chose the Hamburger and Hamilton's stage 29 (HH 29) to investigate the expression of tropomyosin in chick myocardiocytes during 14 days on culture. Throughout 14 days of cell culture, changes in cell morphology were accompanied by a redistribution of tropomyosin in different cell compartments. We used FACScan, SDS-PAGE and densitometric analysis to quantify total cell tropomyosin and concentrations of this protein in different cell fractions. Tropomyosin was found mostly in the cytoskeletal fraction than in the cytoplasmic. When we compared the densitometric values from SDS-PAGE of cells in different stages of development we found that in HH 19, tropomyosin was more abundant in the cytoplasmic than in the cytoskeletal fraction. By HH 29, the two fractions had become inverted, and in HH 39, tropomyosin was clearly more abundant in the cytoskeletal than in the cytoplasmic fraction. In the IFI analysis, tropomyosin was found to label the Stress fiber-like structures (SFL) in different patterns depending on the area of the cell which expressed this protein.

We analyzed the expression profile of isoenzymatic fractions of creatine phosphokinase (EC 2.7.3.2) isotypes MM, MB and BB in three cell lines derived from embryonic rhabdomyosarcomas and a normal counterpart cell line. Electrophoretic data showed that the BB fraction was consistently expressed de novo, in contrast with its counterpart in normal tissue. The BB fraction may serve as new tumoral marker for the diagnosis of rhabdomyosarcoma. In addition, the appearance of macrocreatine kinase type-1 in this type of neoplasm may serve to reinforce the diagnosis when rhabdomyosarcoma is suspected.

We analyzed the distribution and labeling patterns of troponin-T, a protein involved in the regulation of striated muscle contraction, in myocardiocytes obtained from chick embryos in Hamburger and Hamilton's stage 25 and 39, and cultured for 8 days. Troponin-T expression was examined with indirect immunofluorescence, densitometry, fluorescence-activated cell sorting, electrophoresis and immunoblotting. The patterns of expression of troponin-T were compared with those of actin to determine possible correlations in different stages of chick embryo development and culture. Our findings show that in both stages of embryonic development, the cellular accumulation of troponin-T changed after 8 days of culture. Our results revealed a quantitative modification with time: after 4 days of culture there was a significant increase in this protein, followed by a slight additional increase after 8 days of culture. Flow cytometry findings confirmed these trends over time, showing a significant increase in positive cells after 4 days, followed by a smaller rise after 8 days of culture. In comparison with actin, this pattern was similar only in cells from Hamburger and Hamilton's stage 25 embryos.

We used sodium dodecylsulphate polyacrylamide gel electrophoresis and immunoblotting to analyze the effects of the fibric acid derivatives bezafibrate, fenofibrate and gemfibrozil on the accumulation of actin in the cytoplasmic and cytoskeletal fraction of cultured myocardiocytes. All three drugs tested modified cellular and subcellular actin in different ways, and the findings are thought to be related with the secondary effect of arrhythmia known to be caused by these drugs. Bezafibrate and gemfibrozil more markedly affected accumulation of actin by myocytes, while fenofibrate interfered less notably with the accumulation of this protein.

In a total of 496 fetuses and newborns ranging in body weight from 60 to 5000 g, we performed a morphometric study of the ascending aorta, the descending aorta, the aortic isthmus, the right pulmonary artery, the left pulmonary artery and the arterial duct. In all, nine different parameters were measured in each specimen. The variations in the correlations between two measurable characteristics, namely body weight and each of the morphometric parameters, were analyzed. The minimum, normal and maximum patterns of normality for each parameter were obtained with regression equations. We compared statistically the diameter of the arterial duct with the diameter of the ascending and descending aorta, the aortic isthmus, the right pulmonary artery and the left pulmonary artery with one way analysis of variance, using Bonferroni's test in the pairwise comparisons. The diameter of the arterial duct was smaller than the diameter of the ascending and descending aortas, the aortic isthmus, the right pulmonary artery and the left pulmonary artery, and the diameter of the ascending aorta was larger than that of the descending aorta. These morphological data have immediate clinical and surgical applications in the treatment of fetal and perinatal cardiovascular disorders.

We analyzed desmin and vimentin accumulation in chick myocardiocyte cultures treated with the fibric acid derivatives bezafibrate, fenofibrate and gemfibrozil. The most noteworthy finding was the 50% decrease in the cytoplasmic desmin fraction in cells treated with gemfibrozil in comparison to control cultures, and the 19% increase in the cytoskeletal fraction in cultures treated with gemfibrozil and with bezafibrate. Vimentin accumulation by cells treated with bezafibrate was similar to that in control cultures, however the cytoskeletal vimentin fraction rose by 26% after treatment with gemfibrozil, and fell 13% after treatment with fenofibrate. No vimentin was found in the cytoplasmic fraction of cell treated with bezafibrate. Given the role of intermediate filaments in heart muscle contraction, fibric acid derivative- induced changes in the cytoplasmic and cytoskeletal concentrations of intermediate filament proteins may be related with the secondary effects of these drugs on heart rate.

The cytoskeleton of the myocardiocyte is a complex network of protein filaments. The intermediate filaments, consisting fundamentally of desmin, are considered markers of cellular differentiation. We used indirect immunofluorescence with specific monoclonal antibody to characterize the expression of desmin during the different phases of heart maturation, and demonstrated a direct relationship between the expression of this protein and myocardiocyte development. Our results show that in auricular myocardiocytes, desmin is present in early stages of development, whereas in ventricular myocardiocytes, this protein, which is intimately involved in contractile function, first appears in HH stage 25.

We studied the distribution of intermediate filament proteins during several stages of chick embryo heart development by indirect immunofluorescence and fluorescence-activated cell surface analysis. Vimentin is the predominant intermediate filament during the early stages of cardiac genesis, while desmin appears essentially with maturation. Desmin is the main subunit protein of intermediate filaments in the mature myocyte.