Myocardial infarction due to coronary abnormalities in pulmonary atresia with intact ventricular septum.

We describe the clinical course, angiography, and histopathology of a newborn male with pulmonary atresia and intact ventricular septum who succumbed to a myocardial infarction. Angiography demonstrated right ventricular-dependent coronary circulation and focal areas of coronary narrowing. His clinical course was characterized by attacks of sudden irritability, consistent with ischemia. Histology demonstrated significant coronary artery narrowing secondary to fibromuscular dysplasia as well as evidence of new and old infarction. This case illustrates the severity of coronary lesions in pulmonary atresia and the potential for progression of coronary obstruction and insufficiency, and it provides correlation between angiography, ischemic symptoms, and pathology.

Molecular basis of anthracycline-induced cardiotoxicity and its prevention.

Anthracyclines are a class of highly potent antitumor antibiotics utilized against hematologic and solid tumors in children and in adults. Their use has been limited primarily by their cardiotoxic side effects, which may lead to congestive heart failure. Although there is a linear relationship between the cumulative dose received and the incidence of cardiotoxicity, in some patients cardiotoxicity may develop at doses below the generally accepted threshold level. Anthracycline-induced cardiotoxicity is believed to be related to the generation of highly reactive oxygen species, which, by means of membrane lipid peroxidation, cause direct damage to cardiac myocyte membranes. Another important factor may be the relatively poor antioxidant defense system of the heart. In an attempt to circumvent these toxic effects, a wide variety of antioxidants have been used in cell culture, animal, and human studies without consistent beneficial effects. Moreover, none of the agents used to date are designed to act selectively upon the heart. If the cardiac complications resulting from anthracyclines could be reduced and/or prevented, higher doses could potentially be used, thereby increasing cancer cure rates. Furthermore, the incidence of cardiac toxicity resulting in congestive heart failure or even heart transplantation would be reduced, therefore increasing the quality and extent of life for cancer survivors. This article will review the basic science of free radical biology, the biology of oxygen-derived free radicals and antioxidant proteins, and explore some new and innovative approaches to limiting and/or preventing anthracycline-induced cardiotoxicity.

Protein binding by the 3' untranslated region of alpha-striated tropomyosin.

Tropomyosin is a component protein of the thin filament system in striated muscle, regulating the interaction between actin and myosin. The 3' untranslated region of the alpha-striated tropomyosin gene (TM UTR) induces muscle differentiation when expressed in primary fibroblasts, but the mechanism has not been defined. We hypothesize that fibroblasts utilize resident proteins to effect this response, perhaps by TM UTR binding to protein(s). In order to facilitate identification of protein(s) involved in mediating this differentiation response, we investigated the potential for this sequence to bind to cellular protein utilizing electrophoretic mobility gel shifting analysis (EMSA) with and without UV cross-linking. Under very specific conditions (including pH, KCl, and Mg concentration and extent of phosphorylation of protein), the TM UTR is able to bind protein in cells that differentiate upon TM UTR expression. Protein binding is significantly more extensive in cytoplasmic than nuclear protein preparations. Secondary structure of the RNA probe facilitates protein binding. The molecular masses of bound proteins are approximately 42 and 115 kDa under basal conditions. EMSA analysis of extract from cultured skeletal muscle confirms that protein binding by the TM UTR occurs in this cell type, and is more extensive in less differentiated cells. The demonstration of highly regulated protein binding by the TM UTR raises the possibility that this sequence may cause differentiation by binding to endogenous proteins, and further that this sequence may play a role in normal differentiation. Identification of proteins bound by the TM UTR will be necessary to completely define the mechanism by which it causes differentiation.

Misdirected vimentin messenger RNA alters cell morphology and motility.

Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the (beta)-actin 3' untranslated region (3' UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or (beta)-galactosidase that localized through the (beta)-actin 3' UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.

Percutaneous pulmonary artery gene transfer in pigs using naked plasmid DNA delivered during angioplasty.

Gene transfer techniques are increasingly being used to study blood vessel biology and develop models for gene therapy. To date, there are no reports of pulmonary vascular gene transfer performed either without adjunctive agents or during angioplasty. We sought to demonstrate the feasibility of recombinant gene transfer to the pulmonary artery of juvenile pigs using naked plasmid DNA delivered via percutaneous angioplasty techniques. Plasmid DNA directing the expression of beta-galactosidase was used to transfect one pulmonary artery while the contralateral vessel served as an untreated control. One delivery technique used a standard angioplasty balloon coated with a DNA-heparin mixture. The second involved infusion of DNA between an angioplasty balloon and a surrounding, microporous balloon. Vessels were harvested 3 or 4 days following gene delivery. Protein expression was demonstrated by immunohistochemical staining in transfected but not control vessels in 9/9 pigs. Vascular wall expression was limited to endothelial cells. Pulmonary artery gene transfer using naked plasmid DNA delivered via percutaneous angioplasty techniques is feasible. Using naked plasmid DNA removes the potential for toxicity associated with adjunctive agents. The described techniques provide novel methods for studying pulmonary vascular biology in vivo and for developing future gene therapies.

Muscle-specific transcription factors in fibroblasts expressing the alpha-striated tropomyosin 3' untranslated region.

The alpha-striated tropomyosin 3' untranslated region (TM UTR) promotes differentiation of fibroblasts into cells resembling skeletal muscle. To investigate the mechanism of this observation, RNA harvested from transfected primary fibroblasts was used for semiquantitative RT-PCR with primers specific for muscle transcription factors, showing that myoD and myogenin transcripts are detected in these cells, but that differentiation after TM UTR expression is independent of a detectable increase in these transcripts. Double immunofluorescent staining with antibodies to myoD family members and to titin confirms that muscle differentiation in TM UTR-transfected fibroblasts is independent of production of any transcription factor in this family. In contrast, the muscle transcription factor myocyte enhancer factor 2 (mef-2) is strongly expressed after transfection of fibroblasts with the TM UTR. The increase in mef-2 protein is due to an increase in the steady-state level of its mRNA, as shown by Northern analysis. The expression of p21 ordinarily observed in skeletal myogenesis before the expression of muscle-specific proteins is not seen in fibroblasts induced to differentiate by the TM UTR. These results demonstrate that post-transcriptional regulation of myoD family members is seen in fibroblasts, and that the TM UTR induces muscle differentiation independent of the myoD transcription factors and without expressing proteins characteristic of terminal withdrawal from the cell cycle. Finally, an increase in the steady-state level of mef-2 transcripts appears in the proximal pathway of myogenic activation in response to expression of the TM UTR. These results imply that fibroblasts can utilize an additional differentiation route upon TM UTR expression resulting in mature muscle other than that requiring myoD family members.

Assembly of tropomyosin isoforms into the cytoskeleton of avian muscle cells.

Tropomyosin (TM) is a component of microfilaments of most eukaryotic cells. In striated muscle, TM helps confer calcium sensitivity to the actin-myosin interaction. TM is a fibrillar, self-associating protein that binds to the extended actin filament system. We hypothesized that these structural features would permit TM to undergo assembly into the cytoskeleton during translation, or cotranslational assembly. Pulse-chase experiments with [35S]methionine and pulse experiments with [3H]puromycin followed by extraction and immunoprecipitation of TM were performed to examine the mechanism of assembly of TM into the cytoskeleton in cultured avian muscle cells. Pulse-chase experiments provide kinetic evidence for cotranslational assembly of TM in skeletal and cardiac muscle. Demonstration of a large majority of completed TM on purified skeletal muscle microfilaments after a short labeling period confirms that these kinetic data are not related to trapping of TM within the actin network of the cytoskeleton. Nascent TM peptides are demonstrated on the cytoskeleton of muscle cells after a short metabolic pulse followed by puromycin treatment to release nascent peptides from ribosomes or after labeling with [3H]puromycin. Nascent chain localization to the cytoskeleton independent of ribosomal attachment further confirms the high degree of cotranslational assembly of this protein. The extent of cotranslational assembly is similar before and after the formation of significant myofibril in myotubes, suggesting that cotranslational assembly of TM is active during contractile apparatus assembly in muscle differentiation. This is the first report where assembly mechanism has been predicted to be cotranslational based upon structural features of a cytoskeletal protein.

Transdifferentiation of chicken embryonic cells into muscle cells by the 3' untranslated region of muscle tropomyosin.

Transfection with a plasmid encoding the 3' untranslated region (3' UTR) of skeletal muscle tropomyosin induces chicken embryonic fibroblasts to express skeletal tropomyosin. Such cells become spindle shaped, fuse, and express titin, a marker of striated muscle differentiation. Skeletal muscle tropomyosin and titin organize in sarcomeric arrays. When the tropomyosin 3' UTR is expressed in osteoblasts, less skeletal muscle tropomyosin is expressed, and titin expression is delayed. Some transfected osteoblasts become spindle shaped but do not fuse nor organize these proteins into sarcomeres. Transfected cells expressing muscle tropomyosin organize muscle and nonmuscle isoforms into the same structures. Thus, the skeletal muscle tropomyosin 3' UTR induces transdifferentiation into a striated muscle phenotype in a cell-type-specific context.

Specific and quantitative immunoprecipitation of tropomyosin and other cytoskeletal proteins by magnetic separation.

Immunoprecipitation is a powerful technique for purifying many proteins for which specific antibodies exist. Magnetic separation has recently been demonstrated to be effective in the immunoprecipitation of cell-surface proteins. We have used magnetic separation with anti-immunoglobulin or protein A bound to magnetic particles to immunoprecipitate labeled muscle tropomyosin and several other cytoskeletal proteins for which specific antibodies exist. We have not found it necessary to bind antigen-specific antibody to the magnetic particles, increasing the versatility of the technique. The quantitative recovery of tropomyosin from muscle cultures using magnetic separation is superior to Staph A (protein A-positive Staphylococcus aureus cells). The specificity of magnetic separation also compares favorably with Staph A for immunoprecipitation of muscle tropomyosin. Fibroblast tropomyosin, vimentin (from muscle and osteoblast) and myosin heavy chain are other cytoskeletal proteins that are easily recovered with magnetic separation. Magnetic separation, therefore, appears to be a valuable technique for the immunoprecipitation of cytoskeletal proteins from various cell types.

Thin filament changes during in vivo rat heart development.

Developmental differences in myocardial performance are known to exist. It is likely that the profile of protein isoforms present on the developing thin filament contributes to these observed differences. We have prepared thin filaments from developing and mature rat hearts by using an immunoprecipitation procedure developed in our laboratory. Analysis of these isolated thin filaments by Western immunoblots and two-dimensional gel electrophoresis demonstrates troponin I and troponin T isoform switching on the developing thin filament. Troponin I isoform switching begins by embryonic d 18 and is complete before the 3rd postnatal wk. Troponin T isoform switching begins between embryonic d 18 and birth and is complete between the 2nd and 3rd postnatal wk. The degree of phosphorylation of tropomyosin in thin filaments appears to be developmentally regulated, decreasing with advancing age. Nonmuscle isoforms of tropomyosin are also detectable in thin filaments from developing and mature rat hearts. These phenomena (troponin isoform switching, the degree of phosphorylation of tropomyosin, and the presence of nonmuscle isoforms of tropomyosin on cardiac thin filaments) likely play a role in the function of immature thin filaments and in the assembly of mature thin filaments.