Effect of geraniol on rat cardiomyocytes and its potential use as a cardioprotective natural compound.

AIMS: Reactive oxygen species (ROS) are generated in the ischaemic myocardium especially during early reperfusion and affect myocardial function and viability. Monoterpenes have been proposed to play beneficial roles in diverse physiological systems; however, the mechanisms of action remain largely unknown. This study aims to assess the effect of monoterpene geraniol (GOH) on neonatal rat ventricular cardiomyocytes (NRVCs) subjected to oxidative stress. MAIN METHODS: We used an in vitro model of hypoxia-reoxygenation. Cardioprotective (AMPK) and cardiotoxic (ERK1/2, ROS) signaling indicators were measured. Assays were performed by fluorogenic probes, MTT assays and Western-blots. KEY FINDINGS: We determined that the addition of GOH (5-200µM) to cultured normoxic and hypoxic-NRVCs diminished the endogenous production of ROS in stressed cardiomyocytes. We observed that GOH treatment increased pAMPK levels and decreased pERK1/2 levels in cultured NRVCs. SIGNIFICANCE: This report suggests that GOH is a candidate cardioprotective natural compound that operates by blunting the oxidative stress signaling that is normally induced by hypoxia-reoxygenation.

Non-resident stem cell populations in regenerative cardiac medicine.

The adult heart displays a low proliferation capacity, compromising its function if exposed to distinct biological insults. Interestingly, the observation that an increasing number of cell types display an unpredicted cellular plasticity has opened new therapeutical avenues. In this review we will summarize the current knowledge of non-resident stem cells that can be putatively used for cardiac regeneration. At present, bone marrow stem cells have been extensively studied as a cellular source to heal the heart; however, their myocardial contribution is highly limited. Experimental studies have demonstrated that skeletal myoblasts can engraft into the heart, although, unfortunately, they lead to myocardial uncoupling. Embryonic stem cells can spontaneously generate cardiomyocytes that exhibit a variety of electrophysiological phenotypes. Several constrains should nonetheless be overcome before entering the clinical arena, such as the ability to direct and control the generation of cardiomyocytes into a single myocardial lineage.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation.

Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II.

[Developmental genes and heart disease]. / Genes del desarrollo y enfermedad cardíaca.

The past three years can be considered in cardiology as critical for understanding the relevance of developmental genes in the adult cardiac physiology. Also, for the first time, endogenous control of programmed cell death has been demonstrated to mark the transition between normal adaptation and cardiac hypertrophy. Most of this work has been based on previous analysis using molecular markers of cardiac determination and differentiation, work that has served a double aim: First, the determination of the cellular process that contribute to the specification of the working heart and secondly, the characterization of key regulatory factors in cardiogenesis. These studies in conjunction with the recent availability of single gene mutation in transgenic mice have furnished a new perspective in the nature of cardiac defects either in shape or function. Here we review some of the key factors in cardiac morphogenesis from the perspective of the analysis of gene mutation.

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

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

Expression patterns of FHL/SLIM family members suggest important functional roles in skeletal muscle and cardiovascular system.

LIM domain containing proteins play critical roles in animal development and cellular differentiation. Here, we describe the cloning and expression patterns of three members of the four and a half LIM domain-only protein family, FHL1, 2, and 3, from mouse. A comparison of embryonic expression patterns of these three highly-related genes indicates that they are expressed in an overlapping pattern in the developing cardiovascular system, and skeletal muscle. In adult tissues, the three genes are expressed in a predominant and overlapping manner in cardiac and skeletal muscle. Of the three genes, FHL2 appears to have the most restricted expression pattern during development, in heart, blood vessels, and skeletal muscle. Expression in heart is highest in cardiac septa and in the region adjacent to the atrio-ventricular ring, suggesting a potential role in septation or conduction system development. In the heart, FHL1expression was observed strongly in developing outflow tract, and to a lesser extent in myocardium. FHL3 displays low and ubiquitous expression during mouse development. Cardiac ventricular expression of FHL1, but not FHL2 or FHL3, was upregulated in two mouse models of cardiac hypertrophic and dilated cardiomyopathy. Taken together, these data indicate the potential importance of this FHL family in the development and maintenance of the cardiovascular system and striated muscle, and suggest that FHL1 may play a role in the development of heart disease.

Left-right determination.

Recent advances have given us new insights into the molecular basis of organ position. A gene cascade that determines left-right positioning of organ primordia has emerged. In here we present the current knowledge of the molecular determinants of organ positioning during vertebrate embryogenesis.

Deletion of 150 kb in the minimal DiGeorge/velocardiofacial syndrome critical region in mouse.

Deletions or rearrangements of human chromosome 22q11 lead to a variety of related clinical syndromes such as DiGeorge syndrome (DGS) and velo--cardiofacial syndrome (VCFS). In addition, patients with 22q11 deletions have an increased incidence of schizophrenia and several studies have mapped susceptibility loci for schizophrenia to this region. Human molecular genetic studies have so far failed to identify the crucial genes or disruption mechanisms that result in these disorders. We have used gene targeting in the mouse to delete a defined region within the conserved DGS critical region (DGCR) on mouse chromosome 16 to prospectively investigate the role of the mouse DGCR in 22q11 syndromes. The deletion spans a conserved portion ( approximately 150 kb) of the proximal region of the DGCR, containing at least seven genes ( Znf74l, Idd, Tsk1, Tsk2, Es2, Gscl and Ctp ). Mice heterozygous for this deletion display no findings of DGS/VCFS in either inbred or mixed backgrounds. However, heterozygous mice display an increase in prepulse inhibition of the startle response, a manifestation of sensorimotor gating that is reduced in humans with schizophrenia. Homozygous deleted mice die soon after implantation, demonstrating that the deleted region contains genes essential for early post-implantation embryonic development. These results suggest that heterozygous deletion of this portion of the DGCR is sufficient for sensorimotor gating abnormalities, but not sufficient to produce the common features of DGS/VCFS in the mouse.

PPAR gamma is required for placental, cardiac, and adipose tissue development.

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.

Multiple left-right asymmetry defects in Shh(-/-) mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of Lefty-1.

Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in Shh(-/-) embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.

Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis.

Although accumulating evidence suggests that the heart develops in a segmental fashion, the molecular mechanisms that control regional specification of cardiomyocytes in the developing heart remain largely unknown. In this study, we have used the mouse cardiac-restricted ankyrin repeat protein (CARP) gene as a model system to study these mechanisms. The CARP gene encodes a nuclear co-regulator for cardiac gene expression, which lies downstream of the cardiac homeobox gene, Nkx 2.5, and is an early marker of the cardiac muscle cell lineage. We have demonstrated that the expression of the gene is developmentally down regulated and dramatically induced as part of the embryonic gene program during cardiac hypertrophy. Using a lacZ/knock-in mouse and three lines of transgenic mouse harboring various CARP promoter/lacZ reporters, we have identified distinct 5' cis regulatory elements of the gene that can direct heart segment-specific transgene expression, such as atrial versus ventricular and left versus right. Most interestingly, a 213 base pair sequence element of the gene was found to confer conotruncal segment-specific transgene expression. Using the transgene as a conotruncal segment-specific marker, we were able to document the developmental fate of a subset of cardiomyocytes in the conotruncus during cardiogenesis. In addition, we have identified an essential GATA-4 binding site in the proximal upstream regulatory region of the gene and cooperative transcriptional regulation mediated by Nkx2.5 and GATA-4. We have shown that this cooperative regulation is dependent on binding of GATA-4 to its cognate DNA sequence in the promoter, which suggests that Nkx2.5 controls CARP expression, at least in part, through GATA-4.

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.

Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.

p53 is a transcriptional activator of the muscle-specific phosphoglycerate mutase gene and contributes in vivo to the control of its cardiac expression.

The role that the p53 tumor suppressor gene product plays in cellular differentiation remains controversial. However, recent evidence indicates that p53 is required for proper embryogenesis. We have studied the effect of p53 on the expression mediated by the promoter of the rat muscle-specific phosphoglycerate mutase gene (M-PGAM), a marker for cardiac and skeletal muscle differentiation. Experiments involving transient transfection, mobility shift assay, and site-directed mutagenesis demonstrated that p53 specifically binds and transactivates the M-PGAM promoter. The p53-related proteins p51A and p73L also transactivated M-PGAM. Moreover, stable expression of a p53 dominant mutant in C2C12 cells blocked the induction of M-PGAM expression during the myoblast to myotube transition and the ability of p53, p51A, and p73L to transactivate the M-PGAM promoter. In addition, impaired expression of M-PGAM was observed in a subset of p53-null animals in heart and muscle tissues of anterior-ventral location. These results demonstrate that p53 is a transcriptional activator of M-PGAM that contributes in vivo to the control of its cardiac expression. These data support previous findings indicating a role for p53 in cellular differentiation.

Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II.

The embryonic cellular events that set the asymmetry of the genetic control circuit controlling left-right (L-R) axis determination in mammals are poorly understood. New insight into this problem was obtained by analyzing mouse mutants lacking the KIF3A motor subunit of the kinesin-II motor complex. Embryos lacking KIF3A die at 10 days postcoitum, exhibit randomized establishment of L-R asymmetry, and display numerous structural abnormalities. The earliest detectable abnormality in KIF3A mutant embryos is found at day 7.5, where scanning electron microscopy reveals loss of cilia ordinarily present on cells of the wild-type embryonic node, which is thought to play an important role in setting the initial L-R asymmetry. This cellular phenotype is observed before the earliest reported time of asymmetric expression of markers of the L-R signaling pathway. These observations demonstrate that the kinesin-based transport pathway needed for flagellar and ciliary morphogenesis is conserved from Chlamydomonas to mammals and support the view that embryonic cilia play a role in the earliest cellular determinative events establishing L-R asymmetry.

A novel putative protein-tyrosine phosphatase contains a BRO1-like domain and suppresses Ha-ras-mediated transformation.

To investigate a potential role of protein-tyrosine phosphatases (PTPases) in myocardial growth and signaling, a degenerate primer-based reverse transcription-polymerase chain reaction approach was used to isolate cDNAs for proteins that contain a PTPase catalytic domain. Among the 16 cDNA clones isolated by reverse transcription-polymerase chain reaction from total neonatal rat cardiomyocyte RNA, one, designated PTP-TD14, was unique. Subsequent isolation and sequencing of a full-length PTP-TD14 cDNA confirmed that it encodes a novel 164-kDa protein, p164(PTP-TD14). The C-terminal region contains the PTP-like domain, whereas the N-terminal region shows no homology to any known mammalian protein. However, this region is homologous to a yeast protein, BRO1, that is involved in the mitogen-activated protein kinase signaling pathway. Like BRO1, p164(PTP-TD14) contains a proline-rich region with two putative SH3-domain binding sites. By Northern blot analysis, PTP-TD14 is expressed as a 5.3-kilobase pair transcript, not only in neonatal heart but also in many adult rat tissues. When expressed in either COS-7 or NIH-3T3 cells, p164(PTP-TD14) localizes to the cytoplasm in association with vesicle-like structures. Expression of p164(PTP-TD14) in NIH-3T3 cells inhibits Ha-ras-mediated transformation more than 3-fold. This inhibitory activity is localized to the C-terminal PTPase homology domain, since no inhibition of Ha-ras-mediated focus formation was observed with a PTP-TD14 mutant, in which the putative catalytic activity was presumably inactivated by a point mutation. These findings indicate that PTP-TD14 encodes a novel protein that may be critically involved in regulating Ha-ras-dependent cell growth.

Hunting down nucleic acid binding factors in the cardiovascular system.

Transcription regulation of genes active in the cardiovascular system is a complex process, involving DNA and RNA binding proteins. Nucleic acid binding proteins bind to the regulatory DNA and interact with other proteins, including RNA polymerase to initiate and control the level of transcription. The RNA binding proteins have a function in spliceosome formation and in stabilising mRNA. In this review the currently available molecular approaches to analyse regulatory DNA in relation to DNA binding proteins are discussed. Similar techniques that have been developed for RNA binding protein studies are included. In addition to an explanation of the various methods, examples are provided from DNA-protein interactions on genes active in the cardiovascular system, together with strategies for identification and characterisation of new nucleic acid binding proteins active in cardiac or vascular cell types.

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

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

Cloning, mRNA expression, and chromosomal mapping of mouse and human preprocortistatin.

Cortistatin is a 14-residue putative neuropeptide with strong structural similarity to somatostatin and is expressed predominantly in cortical GABAergic interneurons of rats. Administration of cortistatin into the brain ventricles specifically enhances slow-wave sleep, presumably by antagonizing the effects of acetylcholine on cortical excitability. Here we report the identification of cDNAs corresponding to mouse and human preprocortistatin and the mRNA distribution and gene mapping of mouse cortistatin. Analysis of the nucleotide and predicted amino acid sequences from rat and mouse reveals that the 14 C-terminal residues of preprocortistatin, which make up the sequence that is most similar to somatostatin, are conserved between species. Lack of conservation of other dibasic amino acid residues whose cleavage by prohormone convertases would give rise to additional peptides suggests that cortistatin-14 is the only active peptide derived from the precursor. As in the rat, mouse preprocortistatin mRNA is present in GABAergic interneurons in the cerebral cortex and hippocampus. The preprocortistatin gene maps to mouse chromosome 4, in a region showing conserved synteny with human 1p36. The human putative cortistatin peptide has an arginine for lysine substitution, compared to the rat and mouse products, and is N-terminally extended by 3 amino acids.