A novel muscle DNA-binding activity in the GLUT1 promoter.

GLUT1 glucose transporters are highly expressed in proliferating and transformed cells and serum and cAMP or the transcription factor Sp1 induce GLUT1 gene transcription. Here we identified a cis element situated at -46/-37 (MG1E - muscle-specific GLUT1 element) to which muscle-specific nuclear factors bind, and the DNA-protein complexes showed electrophoretic mobility of 41 and 32 kDa. MyoD over-expression induced the generation of MG1E-protein complexes characteristic of myoblast cells. MG1E does not bind any known factors defined in databases. Mutation of the MG1E sequence impaired transcriptional activity of the GLUT1 promoter specifically in skeletal or cardiac muscle cells. The transcriptional activity of the GLUT1 promoter induced by either Sp1, cAMP or serum was markedly reduced when MG1E was inactivated. We propose that the MG1E sequence permits the binding of muscle-specific nuclear factors and a maximal transcriptional activity in muscle cells in response to Sp1, cAMP or serum.

A novel functional co-operation between MyoD, MEF2 and TRalpha1 is sufficient for the induction of GLUT4 gene transcription.

We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.

Overexpression of connexin 43 in skeletal myoblasts: Relevance to cell transplantation to the heart.

OBJECTIVE: Skeletal myoblast transplantation is a promising strategy for treating end-stage heart failure. One potential problem in the development of functional, synchronously contracting grafts is the degree of intercellular communication between grafted myoblasts and host cardiomyocytes. Thus it is expected that enhancement of intercellular gap junction formation would result in improved efficiency of skeletal myoblast transplantation. In this study we investigated whether myoblasts overexpressing connexin 43, a major cardiac gap junction protein, would enhance this intercellular communication. METHODS AND RESULTS: L6 rat skeletal myoblast cell lines overexpressing connexin 43 were generated by means of gene transfection and clonal selection. Connexin 43 overexpression of these myoblasts, which continued both in undifferentiated and differentiated states (up to 17-fold greater protein level in comparison with control-transfected myoblasts, as measured with Western blotting), was observed on cell surfaces where gap junctions should exist. Both dye microinjection and scrape loading with fluorescent dyes showed enhancement in intercellular dye transfer between connexin 43-transfected myoblasts compared with that found in control-transfected cells. Morphologically, these myoblasts fused and differentiated into multinucleated myotubes more rapidly, demonstrating a higher level of cellular creatine kinase activity as a marker of myogenic differentiation throughout the culture period compared with that of control-transfected myoblasts. CONCLUSIONS: We have generated connexin 43-overexpressing skeletal myoblast cell lines that resulted in improved formation of functional intercellular gap junctions, which could be relevant to synchronous contraction of grafted myoblasts in the heart. In addition, these cells demonstrated more rapid differentiation, which would also be advantageous in a graft for transplantation to the heart.

Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter.

OBJECTIVES: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. METHODS: A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. RESULTS: At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. CONCLUSION: The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.

Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart.

BACKGROUND: Graft survival after skeletal myoblast transplantation is affected by various pathological processes caused by environmental stress. Heat shock is known to afford protection of several aspects of cell metabolism and function. We hypothesized that prior heat shock treatment of graft cells would improve their survival after cell transplantation. METHODS AND RESULTS: L6 rat skeletal myoblasts expressing ss-galactosidase (ss-gal) were subjected to heat shock (42 degrees C, 1 hour). Increased expression of heat shock protein 72 was detected 24 hours later in the heat-shocked cells. After hypoxia-reoxygenation in vitro, lactate dehydrogenase leakage was significantly attenuated in the heat-shocked cells; in addition, the percentage of early apoptosis was lower in this group measured by flow cytometry with annexin V staining. For the in vivo study, 1 x 10(6) heat-shocked (hsCTx) or normal-cultured (CTx) myoblasts were infused into the explanted rat hearts through the coronary artery followed by heterotopic heart transplantation. ss-gal activity was significantly higher in the hsCTx group after cell transplantation, with an estimated 8 x 10(6) surviving cells per heart in the hsCTx group and 5 x 10(6) cells in the CTx group on day 28. Discrete loci of grafted cells were globally observed in the myocardium of the hsCTx and CTx groups, with a higher frequency in the hsCTx group. Surviving myoblasts occasionally differentiated into myotubes and had integrated with the native cardiomyocytes. CONCLUSIONS: Heat-shocked skeletal myoblasts demonstrated improved tolerance to hypoxia-reoxygenation insult in vitro and enhanced survival when grafted into the heart. Heat shock treatment could be useful in improving graft cell survival in cell transplantation.

Development of a novel method for cell transplantation through the coronary artery.

BACKGROUND: Cell transplantation is a promising strategy to treat end-stage heart failure. At present, a popular method to deliver cells into the heart is direct intramuscular injection. This method, however, may not be efficient in spreading cells globally into the myocardium. We have developed a novel method for cell transplantation using intracoronary infusion. METHODS AND RESULTS: An L6 rat skeletal muscle cell line expressing ss-galactosidase (ss-gal) was generated by gene transfection and clonal selection. These cells (10(6) in 1 mL medium) were infused into explanted rat hearts through the coronary artery, followed by heterotopic heart transplantation into the abdomen of recipients. Control hearts were infused with cell-free medium. According to ss-gal activity measurements, approximately 5 x 10(5) grafted cells per heart existed on day 3, increasing to 5 x 10(6) on day 28 in the cell-transplanted hearts. At day 28, discrete loci positively stained for ss-gal were observed throughout the cardiac layers of both left and right coronary territories. Some of them differentiated into ss-gal-positive multinucleated myotubes that aligned with the cardiac fiber axis and integrated into the native myocardium, whereas others formed colonies consisting of undifferentiated myoblasts. Connexin 43, a cardiac gap junction protein, was expressed between grafted cells and native cardiomyocytes. No reduction in cardiac function was observed in a Langendorff perfusion system. CONCLUSIONS: We have developed a unique method for efficient cell transplantation based on intracoronary infusion. This method, potentially applicable in the clinical setting during cardiac surgery, could be useful to globally supply cells to the heart.

A strategy for inducing immune tolerance to valve endothelial cells through gene transfer.

BACKGROUND AND AIM OF THE STUDY: We and others have demonstrated an immune response to homograft valve endothelial cells both in vivo and in vitro. Clinically, this is particularly manifest in children. In an attempt to address this problem we have explored a strategy of inducing specific immune tolerance by genetic manipulation of valve endothelial cells. FasL is an inducer of apoptosis; it binds Fas and results in programmed cell death (apoptosis) of Fas-bearing cells such as T lymphocytes. FasL has been shown to be important in the protection of tissue grafts (testis, cornea, kidney and pancreatic islet) from rejection. The ultimate aim of this work is to determine whether the transfection of FasL into human heart valve endothelial cells can hinder immune rejection by induction of apoptosis in T cells. METHODS: The full-length human FasL cDNA was cloned into a mammalian expression vector containing the neomycin resistance marker. The endothelial cell line HMEC-1 was transfected with the plasmid and selected with antibiotic G418. Cultures from positive clones were analyzed by semi-quantitative polymerase chain reaction (PCR) to determine approximate copy numbers of FasL. Reverse transcription (RT)-PCR was carried out to examine the production of mRNA from the construct. Western blot analysis was performed to detect the protein expression. Cytotoxic assays were subsequently performed to detect the FasL function in those transfected cells. RESULTS: High copy number transfected cell lines were produced, and mRNA and protein expression were confirmed. Preliminary results from cytotoxic assays show that transfected cells have enhanced cytotoxicity in comparison with their parent cell line. CONCLUSION: FasL can be overexpressed in endothelial cells and appears to modify the cells' immunological behavior. These findings could have important implications for enhancing homograft valve durability.

Molecular characterization of interstitial cells isolated from human heart valves.

BACKGROUND AND AIM OF THE STUDY: Myofibroblasts have been described as possessing certain characteristics of both fibroblasts and skeletal myocytes. These cells are of mesenchymal origin, were first described in wound healing, and have been found in many tissues. Myofibroblasts from other tissues have been shown to contract and to express sarcomeric (muscle) genes. In addition, these cells express certain regulatory (transcription factor) genes. The specific alignment of the cells may, at least in part, be governed by tissue polarity signals transmitted by members of the frizzled family of vertebrate tissue polarity genes. The aim of the present study was to characterize interstitial cells, with regard to the expression of myofibroblasts markers, isolated from the human heart valves. The expression of muscle structural, regulatory and tissue polarity genes has been undertaken with a view to understanding the development and contribution of interstitial cells to valve function and structure. METHODS: Interstitial cells were isolated and cultured from aortic, pulmonary, tricuspid and mitral valves of recipient hearts obtained during transplantation. Specific oligonucleotide primer pairs suitable for polymerase chain reaction (PCR) were designed for the genes of interest. Total RNA was extracted from the cultured cells and reverse transcriptase-PCR was used to determine gene expression. RESULTS: Cells from the four valve types were found to express various muscle structural genes. These include the thin filament sarcomeric genes for the cardiac isoforms of troponin T, I and C. Evidence was also found for expression of beta-myosin heavy chain (beta-MHC), alpha-MHC and cardiac myosin light chain 2 (MLC2) in these cells. The tissue polarity genes frizzled 2 (fz2) were expressed in all four valve types analyzed. CONCLUSION: Interstitial cells express a number of genes whose products may have functional significance for heart valves. These include members of the contractile apparatus such as MHC and troponins. The presence of members of the frizzled family, which specify the orientation of cell polarization, in these cells could indicate that interstitial cells are not randomly arranged in the valve tissue. Therefore, interstitial cells isolated from the human heart valves express a number of functionally important genes, suggesting a role in their specialized function.

Expression of 5-hydroxytryptamine receptor subtype messenger RNA in interstitial cells from human heart valves.

BACKGROUND AND AIM OF THE STUDY: Severe heart valve disorder has been reported in patients receiving a combination of the anorectic drugs fenfluramine and phentermine. The exact molecular mechanisms involved remain unknown. Fenfluramine alters the serotonin level in the brain, while phentermine interferes with the pulmonary clearance of serotonin; these data suggest that serotonin levels affect regulation of valve function. The aim of the present study was to characterize the serotonin receptor (5-hydroxytryptamine) subtypes expressed in the interstitial cells of human heart valves. METHODS: Interstitial cells were isolated and cultured from the aortic, pulmonary, mitral and tricuspid valves of recipient hearts obtained during transplantation. Total RNA was extracted from cultured cells in order to determine gene expression by reverse transcription-polymerase chain reaction (RT-PCR) using 5-hydroxytryptamine (5-HT) subtype-specific primer pairs. RESULTS: The results show that: (i) 5-HT 1B and 1D receptor subtypes are expressed in all four heart valves. This is significant as the 1B and 1D receptor subfamilies are the target of the anti-migraine drug sumatriptan, and these receptors regulate cardiac function and movement; (ii) 5-HT 1A, 5-HT 1E and 5-HT 1F are not expressed in interstitial cells isolated from the valves. CONCLUSION: We conclude that preliminary evidence exists for the presence of distinct subsets of 5-HT receptors in human heart valves, indicating that interstitial cells of the valves potentially respond to serotonin levels.

Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter.

The human cardiac troponin I (TnIc) gene exhibits both cardiac-specific and developmentally regulated expression. The structure and expression of this gene as well as the identification of putative regulatory elements have been described previously. This study shows that a minimal promoter containing 98 bp of sequence is sufficient to drive transcription in neonatal rat cardiac myocytes. This region contains several putative cis -regulatory elements including an Initiator element surrounding the start site of transcription, an A/T-rich (TATA/MEF-2) element, two GATA elements and a cytosine-rich region containing overlapping CACC box and Sp1 elements. Using electrophoretic mobility shift assays (EMSAs) this study demonstrates the binding of MEF-2, Oct-1, and recombinant TBP to the A/T-rich element and of GATA-4 to both GATA elements. The CACC/Sp element binds the zinc finger transcription factors Sp1 and Sp3 in addition to an unidentified complex present in neonatal rat cardiac myocytes. Mutation of each of these sites has a deleterious effect on promoter activity as assayed by transient transfection into cardiac myocytes. The data suggest that transcriptional activity of the human TnIc gene can be driven by a compact promoter region and that within this region GATA, MEF-2 Sp1 and CACC box-binding factors are required for optimal activity. Furthermore, a comparison with data obtained for identical elements in the promoters of rodent TnIc genes identifies differences between species which may be of consequence for species-specific promoter function.

Factors involved in GLUT-1 glucose transporter gene transcription in cardiac muscle.

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal.

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T.

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.

Novel BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene.

BTB/POZ-domain C2H2 zinc(Zn)-finger proteins are encoded by a subfamily of genes related to the Drosophila gap gene krüppel. To date, two such proteins, PLZF and LAZ-3/BCL-6, have been implicated in oncogenesis. We have now identified a new member of this gene subfamily which encodes a 62 kDa Zn-finger protein, termed LRF, with a BTB/POZ domain highly similar to that of PLZF. Both human and mouse LRF genes, which localized to syntenic chromosomal regions (19p13.3 and 10B5.3, respectively), were widely expressed in adult tissues and cell lines. At approximately 9.5-10.0 days of embryonic development, the mouse LRF gene was expressed in the limb buds, pharyngeal arches, tail bud, placenta and neural tube. The LRF protein associated in vivo with LAZ-3/BCL-6, but not with PLZF to which it was more related. Although the LRF, or LAZ-3/BCL-6, BTB/POZ domain could readily homodimerize, no heterodimerization was detected in vivo between the LRF and LAZ-3/BCL-6 BTB/POZ domains and interaction between full length LRF and LAZ-3/BCL-6 required the presence of both the BTB/POZ domain and Zn-fingers in each partner protein. As expected from the above results, LRF and LAZ-3/BCL-6 also colocalized with each other in the nucleus. Taken together, our findings suggest that BTB/ POZ-domain Zn-finger proteins may function as homo and heterodimeric complexes whose formation, and hence the resultant effect on transcription of their downstream target genes, is determined by the levels and expression domains of a given partner protein.

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts.

BACKGROUND: Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. METHODS: MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. RESULTS: Morphometric analysis showed that 66.3% +/- 3% to 69.6% +/- 6% of cells in group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta significantly inhibited gene transfer, whereas insulin-like growth factor-I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. CONCLUSION: MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.

Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level.

The protein kinase C (PKC) isoenzymes expressed by human peripheral lung and tracheal smooth muscle resected from individuals undergoing heart-lung transplantation were identified at the protein and mRNA level. Western immunoblot analyses of human lung identified multiple PKC isoenzymes that were differentially distributed between the soluble and particulate fraction. Thus, PKC alpha, PKC betaII, PKC epsilon, and PKC zeta were recovered predominantly in the soluble fraction whereas the eta isoform was membrane-associated together with trace amounts of PKC alpha and PKC epsilon. PKC beta1-like immunoreactivity was occasionally seen although the intensity of the band was uniformly weak. Immunoreactive bands corresponding to PKCs gamma, delta, or theta were never detected. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracted from human lung using oligonucleotide primer pairs that recognise unique sequences in each of the PKC genes amplified cDNA fragments that corresponded to the predicted sizes of PKC alpha, PKC betaI, PKC betaII, PKC epsilon, PKC zeta, and PKC eta (consistent with the expression of PKC isoenzyme protein) and, in addition, mRNA for PKC delta; PCR fragments of the expected size for the supposedly muscle-specific isoform, PKC theta, or the atypical isoenzyme, PKC lambda, were never obtained. The complement and distribution of PKC isoforms in human trachealis were similar, but not identical, to human lung. Thus, immunoreactive bands corresponding to the alpha, betaI, betaII, epsilon, and zeta isoenzymes of PKC were routinely labelled in the cytosolic fraction. In the particulate material PKC alpha, PKC epsilon, PKC alpha, PKC eta, and PKC mu were detected by immunoblotting. With the exception of PKC zeta, RT-PCR analyses confirmed the expression of the PKC isoforms detected at the protein level and, in addition, identified mRNA for PKC delta. Collectively, these data clearly demonstrate the expression of multiple PKC isoenzymes in human lung and tracheal smooth muscle, suggesting that they subserve diverse multifunctional roles in these tissues.

Myocyte enhancer factor 2 (MEF2).

The Myocyte Enhancer Factor 2 (MEF2) proteins are transcription factors expressed during development of all three muscle lineages. Of the four mammalian mef2 genes, three (A, C and D) can be alternatively spliced, producing transcripts and proteins which may have significant functional differences. Specific binding sites for MEF2 proteins have been characterized in many striated muscle genes and MEF2 proteins can trans-activate gene expression both as homo- and heterodimers. Loss-of-function mutants in Drosophila indicate that MEF2 is an essential co-factor, but not a primary determinent, in the development of all three muscle lineages in the fly. Recent data suggest an interaction between the DNA-binding domains of mammalian MEF2 proteins and those of tissue-specific basic helix-loop-helix (bHLH) factors and thyroid hormone receptor alpha 1 (TR alpha 1) in the expression of target genes and the development of specific cell phenotypes. Understanding how MEF2 proteins function in the three mammalian muscle types may allow the development of therapeutic strategies for manipulating muscle growth and characteristics.

Localization of the fast skeletal muscle troponin I gene (TNNI2) to 11p15.5: genes for troponin I and T are organized in pairs.

We have localized the gene encoding the fast skeletal muscle isoform of troponin I (TNNI2) to 11p15.5 by PCR-based analysis of somatic cell hybrid panels: based on the Genebridge4 radiation hybrid panel, TNNI2 is coincident with the marker D11S922. The gene encoding the fast skeletal muscle troponin T gene (TNNT3) has been previously assigned to 11p15.5 suggesting that TNNI2 and TNNT3 may be closely linked. The overall location of genes encoding troponin I and T isoforms now reveals that they are organized at three loci each containing a troponin I/troponin T gene pair. This organization contrasts with all other sarcomeric protein genes and has implications for the evolution of these two gene families, for their regulation and for the analysis of mutations suspected to result in cardiomyopathy.

Isolation and characterization of the human cardiac troponin I gene (TNNI3).

Troponin I (TnI) is a constituent protein of the troponin complex located on the thin filament of striated muscle that provides a calcium-sensitive switch for striated muscle contraction. Unlike other contractile proteins, the cardiac isoform of troponin I (TnIc) is expressed only in cardiac muscle and therefore offers a model for cardiac-specific expression. It is also subject to developmental regulation with increased expression occurring at the time of birth. Here we describe the isolation and characterization of the human TnIc gene (HGMW-approved symbol TNNI3) and its promoter. The gene comprises eight exons contained within 6.2 kb of genomic DNA. The proximal promoter and 1.1-kb 5'-flanking region were sequenced, and several putative cis-acting elements that are conserved between the human and the mouse TnIc genes were identified. In addition, multiple copies of a 37-bp chromosome 19-specific mini-satellite sequence were identified within this region. Following transfection, 2300 bp of 5' sequence is active in both cardiac myocytes and skeletal muscle cells but is inactive in fibroblasts, indicating that it can drive expression but is insufficient to confer cardiac specificity.

The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes.

Primary rat cardiocytes were subjected to either thermal "preconditioning" for 30 min at 43 degrees C or 20 min metabolic "preconditioning" (10 mM deoxyglucose, 20 mM lactate, pH 6.5). Eighteen hours later cells were analysed either for hsp 70i expression or subjected to a subsequent lethal heat stress or simulated ischaemia (10 mM deoxyglucose, 20 mM lactate, 0.75 mM sodium dithionite, 12 mM potassium chloride, pH 6.5) for 2 hours and assessed for survival by trypan blue exclusion. Hsp 70i was induced over 100 fold by thermal "preconditioning" and 30 fold by metabolic "preconditioning" (p < 0.001, p < 0.05), hsp 90 was induced 2.71 fold and 2.24 fold (p < 0.001, p < 0.001) by thermal and metabolic "preconditioning" respectively, while hsp 60 was no induced by either treatment. Preconditioned cultures had improved survival against subsequent lethal heat stress or simulated ischaemia: Thermal "preconditioning" reduced death from 69.22% to 52.46% upon subsequent "lethal" heat stress and from 49.13% to 36.66% upon subsequent "lethal" simulated ischaemia. Metabolic "preconditioning" reduced cell death from 51.29% to 33.8% against subsequent "lethal" heat stress, and from 69.09% to 55.61% upon subsequent "lethal" simulated ischaemia. A second marker of cell death, the release of lactate dehydrogenase activity into the culture media, was reduced to 65% and 60% of control values for thermally preconditioned cells subjected to "lethal" heat or "lethal" simulated ischaemia respectively. Metabolically "preconditioned" cells demonstrated lactate dehydrogenase activity of 59% and 51% that of control values, when subjected to "lethal" heat or "lethal" simulated "ischaemia" respectively.