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Biomedical subjects

E Kardami

Publications and source records attributed to E Kardami.

At least 37 records · Page 2Linked to original sources
PubMed1995

Effect of "enhancer" sequences on ventricular myosin light chain-2 promoter activity in heart muscle and nonmuscle cells.

Positive (HF-1 and HF-2) and negative (HF-3) elements responsible for cardiac-specificity of the rat ventricular myosin light chain-2 (MLC-2v) promoter are contained in a 250 base pair region. The effect of the simian virus 40 (SV40) enhancer or 3 copies of the HF-1, HF-2 and HF-3 elements on MLC-2v promoter activity and specificity was assessed in neonatal rat cardiac myocytes and cardiac nonmuscle cells as well as rat heart myoblast H9c2 and glioma (nonmuscle) C6 cell lines. The SV40 enhancer increased promoter activity by at least 10-fold in both muscle and nonmuscle cell types; however, there was a decrease in cardiac ventricular myocyte-specificity. In contrast, the 3 copies of HF-1, HF-2 and HF-3 elements stimulated MLC-2v promoter activity approximately 3-fold in neonatal ventricular cardiac myocytes alone and, effectively, displayed about a 5-fold increase in specificity over the wild type MLC-2v promoter.

Animals↗
PubMed1995

Characterization of fibroblast growth factor receptor 1 RNA expression in the embryonic mouse heart.

We used reverse transcriptase-polymerase chain reaction (RT-PCR) to clone fibroblast growth factor receptor (FGFR) 1 isoforms from embryonic mouse heart and as a more sensitive method to characterize FGFR1 RNA expression in embryonic and adult mouse hearts. We describe the cloning of both full-length short (2259 base pairs) and long (2526 base pairs) FGFR1 isoform cDNAs which generated 86 and 102 kilodalton proteins, respectively, following in vitro translation. An assessment of FGFR1 RNA indicates that FGFR1-IIIc is the major form in both the embryonic and adult heart but there is an approximately 8.5-fold decrease in RNA levels in the adult. Differential RNA blotting as well as RT-PCR analyses are consistent with a switch in the relative expression of the short versus long FGFR1 isoforms during heart development. The long isoforms are more abundant in the embryo and the short isoforms predominate in the adult. This may be important in the regulation of growth and development of the heart.

Amino Acid Sequence↗
PubMed1995

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury.

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8 +/- 1.5% and 96.5 +/- 3.5% of preischemic levels in control and bFGF-treated hearts (10 micrograms/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 micrograms bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.

Animals↗
PubMed1995

Basic fibroblast growth factor stimulates connexin-43 expression and intercellular communication of cardiac fibroblasts.

Gap junctions (GJ) are membrane specializations responsible for intercellular communication and for ensuring electrical and/or metabolic coupling between cells. They are composed of connexins, a family of related proteins. Connexin-43 (Cx43) is a major connexin of the rat heart, expressed by myocytes as well as non-muscle cells. In this communication we have examined expression of Cx43 by cardiac fibroblasts and regulation of its expression by an endogenous mitogen, basic fibroblast growth factor (bFGF). Recombinant human bFGF, administered to cultured cells which had been maintained in 0.5% serum for 48 h, induced dose-dependent and statistically significant increases in Cx43 mRNA as well as protein accumulation, at 6 h after addition. Intercellular communication was also increased at 6 h but not 30 min after bFGF treatment, as assessed using a scrape-loading protocol. It is concluded that the bFGF-induced stimulation of Cx43 expression caused increased coupling between cardiac fibroblasts. This would be of importance in injured myocardium, the increased bFGF content of which might stimulate electrical coupling involving fibroblasts of the scar tissue.

Animals↗
PubMed1995

Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor.

As a first step in addressing the question of function for basic fibroblast growth factor (bFGF) in the adult myocardium, expression of bFGF receptors by adult rat myocytes was investigated. Cross-linking of 125I-labeled bFGF to purified sarcolemmal vesicles from adult hearts indicated specific binding to 90- to 104-kDa proteins, whereas equilibrium binding studies revealed the presence of "low"-affinity (1 nM) and "high"-affinity (115 pM) sites. Adult myocytes were found to express short and long variants of bFGF receptor 1 (FGFR-1, tyrosine kinase) mRNA. Adult heart overall levels of FGFR-1 mRNA were decreased by about one-third of corresponding fetal values. Several lines of evidence indicated that bFGF receptors in adult cardiomyocytes in situ and/or in isolation are functional. Isolated adult myocytes were found to be capable of heparin-resistant internalization of 125I-labeled bFGF, to lose their viability after interaction with bFGF-saporin (a mitotoxin known to kill cells after entry via the bFGF receptor), and to respond to bFGF by activation of mitogen-activated protein kinase. In addition, introduction of exogenous bFGF into the myocardium by Langendorff perfusion resulted in stimulation of tyrosine phosphorylation in association with cardiomyocyte intercalated disks, as assessed by immunofluorescence. It is concluded that adult cardiomyocytes express functionally coupled high-affinity bFGF receptors and that they are capable of a biologic response to bFGF in vivo.

Animals↗
PubMed1994

The effects of hyperthyroidism on muscular dystrophy in the mdx mouse: greater dystrophy in cardiac and soleus muscle.

Muscle damage and repair were studied in mdx mice treated with triiodothyronine (T3) for 14 days. Hindlimb and cardiac muscles were examined for the severity of dystrophy, the degree of muscle centronucleation, and fiber size. In control and mdx mice, cardiac hypertrophy and skeletal muscle atrophy were present after T3 treatment. Both cardiac and soleus (but not fast-twitch) muscles had larger, more frequent dystrophic lesions in T3-treated mdx mice, and mdx soleus had an increased area of new myotubes after T3. Skeletal myogenesis in mdx mice may have been delayed by excess T3, possibly related to the general reduction in staining for basic fibroblast growth factor in hyperthyroid mice. These are the first observations of a metabolic perturbation which worsens mdx dystrophy and possibly repair in a muscle-specific manner, and are likely related to T3-induced changes in myosin heavy chain expression, and to increased mechanical strain on dystrophin-deficient muscles.

Animals↗
PubMed1994

Over-expression of CUG- or AUG-initiated forms of basic fibroblast growth factor in cardiac myocytes results in similar effects on mitosis and protein synthesis but distinct nuclear morphologies.

Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic fibroblast growth factor (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (22/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of 22/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte cultures was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocyte cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of 22/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte cultures over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of fibroblast growth factor receptor-1 isoforms in the mouse heart: evidence for isoform switching during heart development.

Basic (b) fibroblast growth factor (FGF) mediates various biological responses including mitogenesis and angiogenesis by binding to specific cell surface receptors of the tyrosine kinase family. The bFGF receptor-1 FGFR1) exists in short and long isoforms due to alternate RNA splicing. Minor alterations in the amino acid sequence have also led to reports of different FGFR1 isoforms in different tissues even in the same species. In the absence of any sequence for heart FGFR1 and accumulating evidence for a role of bFGF in heart growth and differentiation, we cloned FGFR1 from embryonic mouse hearts. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate full-length short (2259 base pairs) and long (2526 base pairs) forms of FGFR1 cDNAs which generated 86 and 102 kDa proteins, respectively, following in vitro translation. Embryonic mouse heart FGFR1 differed by seven amino acids from the reported sequence for mouse neuroepithelial FGFR1 and appeared more similar to human placental FGFR1. A single FGFR1 transcript of approximately 4.3 kb was seen in RNA isolated from embryonic as well as adult mouse hearts. There was a decrease (approximately 8.5-fold) in FGFR1 RNA levels in the adult. The majority of FGFR1 transcripts in the adult as well as embryonic heart contained exon IIIc (FGFR1-IIIc) which is associated with isoforms that display the highest affinity for bFGF. However, the relative ratio of short versus long FGFR1 RNA expression was 0.5 in the embryonic heart compared to 5.9 in the adult heart. These results indicate that: (i) structurally distinct short and long FGFR1 isoform RNAs are expressed in the embryonic and adult heart; (ii) FGFR1-IIIc is the major form of receptor expressed in the embryonic as well as adult heart; (iii) the transition from the embryo to the adult stage is associated with a decrease but not absence of FGFR1 RNA expression; and (iv) long FGFR1-isoforms are more abundant in the embryo while short FGFR1 isoforms predominate in the adult.

Amino Acid Sequence↗
PubMed1994

The pituitary-muscle axis in mdx dystrophic mice.

As myogenesis, muscle growth and differentiation and growth factor expression are influenced by thyroid and growth hormone (GH) levels, it is important to investigate the possibility that altered activity of the pituitary-muscle axis prevents the lethal progression of mdx dystrophy and/or contributes to the muscle fiber hypertrophy of limb muscles. The ultrastructure of pituitary and thyroid tissues in age-matched control and mdx mice at 2 and 12 months of age was examined. Pituitary GH, and serum thyroid stimulating hormone (TSH), thyroid hormone (T4), and creatine kinase (CK) levels were measured. Mdx thyroid gland structure was similar to age-matched control glands. Mdx thyroid gland weighed significantly more than in age-matched controls, but was unchanged relative to body weight. TSH and T4 levels were not different from levels in control mice. High CK levels reflected the active dystrophy in mdx muscles. Somatotrophs in mdx pituitaries were hypertrophied in comparison to controls, indicating increased secretory activity, and pituitary GH was slightly but significantly greater in old mdx female mice compared to age-matched female controls. These observations rule out hypopituitary or hypothyroid function as a reason for the low impact of dystrophin deficiency in mdx muscles. Results suggest a contribution by raised GH to the fiber hypertrophy in mdx limb and heart muscle, which might also assist the large capacity for limb muscle regeneration in mdx mice.

Aging↗
PubMed1993

Perinatal phenotype and hypothyroidism are associated with elevated levels of 21.5- to 22-kDa basic fibroblast growth factor in cardiac ventricles.

Control of cardiomyocyte growth and differentiation may be exercised, in part, at the level of expression of endogenous growth factors such as bFGF (basic fibroblast growth factor), believed to act locally, in an autocrine or paracrine fashion. Examination of bFGF accumulation by extract fractionation and immunoblotting indicated a 4-fold increase of an 18-kDa bFGF in adult compared to newborn rat heart ventricles. In contrast, a 22-kDa bFGF species, found to be the predominant form in newborn ventricles, displayed a 2.5-fold decrease in extracts from adult (compared to neonatal) ventricles. Since newborn rats are physiologically hypothyroid, the effect of thyroid status on the accumulation of 22-kDa bFGF in the heart was examined. A 21.5- to 22-kDa bFGF showed a 5-fold increase in extracts from hyperthyroid rat heart ventricles, compared to those from euthyroid controls. The 21.5- to 22-kDa bFGF was essentially unaffected by thyroid status in extracts from brain. Our data point to a correlation between immature or differentiated cardiac phenotype and the predominance of 21- to 22.5-kDa or 18-kDa bFGF species, respectively. Elevated levels of 21.5- to 22-kDa bFGF detected in cardiac ventricles in either physiological or experimentally induced hypothyroidism indicates that thyroid hormone may, directly or indirectly, down-regulate accumulation of the higher molecular mass forms of bFGF in the heart.

Animals↗
PubMed1993

Increased basic fibroblast growth factor (bFGF) accumulation and distinct patterns of localization in isoproterenol-induced cardiomyocyte injury.

Basic FGF is a multifunctional protein which promotes regeneration in several tissues. To investigate involvement in cardiac injury-repair, bFGF accumulation and localization was examined in hearts of rats injected with a single high dose of isoproterenol. The bFGF content of cardiac extracts was analyzed at 6 and 24 hours as well as 1, 4, and 6 weeks by western blotting of heparin-sepharose-bound fractions. The 18 kilodalton bFGF species showed an approximately 2-fold increase in extracts from treated animals compared to non-treated controls. A transient rise in a 21-23 kilodalton bFGF species was seen at 24 hours after treatment. An induction of bFGF mRNA was also observed in treated animals. To localize bFGF in vivo, immunofluorescent labelling with specific antibodies was used at 4-24 hours and 1-4 weeks after treatment. Simultaneous labelling for the cytoskeletal proteins vinculin or vimentin was employed to identify viable myocytes or non-muscle interstitial cells, respectively. Necrotic myocytes, identified by loss of vinculin, displayed a pronounced increase in cytoplasmic anti-bFGF staining compared to adjacent normal myocytes. This increase occurred prior to and may play a role in promoting mobile cell migration and proliferation in areas of necrosis. Viable cardiomyocytes adjacent to fibrotic regions displayed strong pericellular anti-bFGF staining and, occasionally, were also stained by anti-vimentin antibodies, suggesting reexpression of an embryonic phenotype and thus an attempt for regeneration. These data showing increased accumulation and distinct patterns of localization of bFGF in the hearts of isoproterenol-treated animals suggest that this growth factor plays a role in short-term as well as long term response of the myocardium to injury.

Animals↗
PubMed1992

Identification of basic fibroblast growth factor-like proteins in African trypanosomes and Leishmania.

Basic fibroblast growth factor (bFGF) is a multifunctional, heparin-binding, mitogenic polypeptide found in all tissues or cells of multicellular organisms so far examined. Here we report that Trypanosoma brucei rhodesiense procyclic culture forms (PCF) and Leishmania donovani promastigotes grown in serum-containing and serum-free medium, contained peptides of 15-34 kDa which bound heparin-sepharose with high affinity and which reacted in immunoblots with several preparations of antibodies specific for bovine brain bFGF. Similar peptides were not detectable in foetal bovine serum. Immunofluorescence studies showed bFGF-like molecules to have a cytoplasmic distribution in both species growing in serum-free media. A nuclear and/or perinuclear distribution of immunoreactivity was also observed in parasites which had been grown in the presence of serum. The data indicate that both species of parasites synthesize their own bFGF-like molecules. Association of an ubiquitous growth factor with parasitic protozoa may play an important role in parasite multiplication and in host-parasite interactions.

Animals↗
PubMed1991

Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions.

Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.

Animals↗
PubMed1991

Basic fibroblast growth factor in rat brain: localization to glial gap junctions correlates with connexin43 distribution.

Light and electron microscope procedures and antibodies against basic fibroblast growth factor (bFGF) were used to study the immunohistochemical localization of bFGF in rat brain. Throughout all areas of the brain analyzed by LM including grey matter, white matter, ependyma, and leptomeninges bFGF-immunoreactivity consisted of punctate immunolabelling that had an appearance and heterogenous distribution nearly identical to that displayed by the gap junction protein connexin43. By immuno-EM, bFGF was localized to gap junctions between astrocytes. It appears that there is a physical association of bFGF with gap junctions composed of connexin43 and it is suggested that bFGF may exert a regulatory influence on intercellular communication at such junctions.

Animals↗
PubMed1991

Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles.

Mdx mice uniquely recover from degenerative dystrophic lesions by an intense myoproliferative (regenerative) response. To investigate a potential role of endogenous basic fibroblast growth factor (bFGF) in injury-repair processes, we investigated its localization in several striated muscles of mdx and control mice using immunofluorescence labeling with specific antibodies. Basic FGF was localized consistently to the myofiber periphery and nuclei of intact myofibers, as well as in single, dystrophin-positive cells in close association with the myofibers (potential myosatellite cells). In mdx mice, actively degenerating skeletal or cardiac muscle fibers presented intense cytoplasmic anti-bFGF staining prior to mononuclear infiltration. Small regenerating fibers in mdx skeletal muscle exhibited greater bFGF accumulation than adjacent larger myofibers. Strong nuclear anti-bFGF immunolabeling was frequently observed in mdx cardiac myocytes at the borders of necrotic regions. In agreement with differences in intensity of immunolabeling, extracts from slow-twitch muscles contained higher levels of bFGF compared to those from fast-twitch muscles, in both control and mdx mice. In addition, bFGF levels were consistently higher in extracts from all mdx tissues compared to those derived from their control counterparts. Our data suggest that bFGF participates in the degenerative and regenerative responses of striated muscle to dystrophic injury and also indicate a potential involvement of this factor with the physiology of different striated muscles.

Animals↗
PubMed1991

Basic fibroblast growth factor in cultured cardiac myocytes.

Distribution of basic-fibroblast-growth-factor-like peptides in immature cultured cardiac myocytes was investigated using specific antisera and immunolocalization. Basic FGF was detected in association with the external surface of the cell membrane, with specialized intercellular junctions and with the myofibril Z lines in the cytoplasm. Intense, punctate nuclear anti-bFGF labeling was observed in a fraction of interphase myocytes of near-confluent, proliferating cultures. This staining pattern persisted even after the dissolution of the nuclear envelope in prophase myocytes. The pattern of cellular localization of bFGF indicates a continuous participation of this factor in myocyte physiology as well as a role in the cell cycle. Furthermore, the identification of bFGF not only in cell lysates but also in culture media after gentle mechanical disruption suggests that cardiac myocytes may release bFGF in vivo following tissue damage.

Animals↗
PubMed1990

Calcium protects pituitary basic fibroblast growth factors from limited proteolysis by co-purifying proteases.

Extracts from bovine pituitaries and other tissues contained basic fibroblast growth factor-like peptides of 22-26 kda, co-fractionating with smaller, 16-20 kda bFGFs. Heparin-bound, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by tryptic proteolysis. In solution, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by an activity present in pituitary extracts. Calcium protected higher molecular weight pituitary bFGFs from truncation by the endogenous activity, which was not acid-activated, co-purified with bFGF during heparin-sepharose chromatography, remained operant at high salt concentrations and was inhibited by phenylmethan-sulfonyl fluoride.

Animals↗