Cardiac Myosin Light Chain Research Articles

Effects of calmodulin and okadaic acid on myofibrillar Ca2+ sensitivity in cardiac myocytes.

Whereas it has been established that the phosphorylation of 20 kD regulatory myosin light chain (MLC20) is a key regulator of contraction in smooth muscle, troponin complex has been thought to be that of myofibrillar Ca2+ sensitivity in cardiac muscle. To elucidate the role of the phosphorylation of cardiac regulatory myosin light chain (MLC2) in the regulation of cardiac muscle contraction, we observed effects of calmodulin and okadaic acid, a protein phosphatase inhibitor, on myofibrillar Ca2+ sensitivity as estimated by pCa50 values obtained from pCa-tension relationships using beta-escin-skinned cardiomyocytes from Wistar rat hearts, in relation to changes in the phosphorylation of myofibrillar regulatory proteins. Whereas myofibrillar Ca2+ sensitivity tended to be progressively decreased by repeated Ca2+-activation in the absence of calmodulin (pCa50; from 5.91 to 5.86, n = 5), calmodulin (2.5 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 microM enhanced Ca2+-activated force, which was inhibited by 50 microM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.96 to 6.11, n = 6, p < 0.05). Whereas the phosphorylation level of troponin I was not changed by 3 microM okadaic acid, that of MLC2 was significantly increased by the same dose of okadaic acid (from 12 to 31%, n = 4, p < 0.05). These results suggest that MLC2 phosphorylation plays a partial role in the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle.

Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain

The functional significance of the actin binding region at the amino terminus of the cardiac essential myosin light chain (ELC) remains obscure. Previous experiments carried out in vitro indicated that modulation of residues 5-14 could induce an inotropic effect, increasing maximal ATPase activity at submaximal Ca(2+) concentrations (Rarick, H. M., Opgenorth, T. J., von Geldern, T. W., Wu-Wong, J. R., and Solaro, R. J. (1996) J. Biol. Chem. 271, 27039-27043). Using transgenesis, we effected a cardiac-specific replacement of ELC with a protein containing a 10-amino acid deletion at positions 5-14. Both the ventricular (ELC1vDelta5-14) and atrial (ELC1aDelta5-14) isoforms lacking this peptide were stably incorporated into the sarcomere at high efficiencies. Surprisingly when the kinetics of skinned fibers isolated from the ELC1vDelta5-14 or ELC1aDelta5-14 mice were examined, no alterations in either unloaded shortening or maximum shortening velocities were apparent. Myofibrillar Mg(2+)-ATPase activity was also unchanged in these preparations. No significant changes in the fiber kinetics in the cognate compartments were observed when either deletion-containing protein replaced endogenous ELC1v or ELC1a. The data indicate that the previously postulated importance of this region in mediating critical protein interactions between the cardiac ELCs and the carboxyl-terminal residues of actin in vivo should be reassessed.

Identification of a Novel Human Ankyrin-Repeated Protein Homologous to CARP

We cloned a novel ankyrin repeat protein, Arpp, by immunoscreening a cDNA library constructed from a human esophageal carcinoma cell line, TE1, with an antibody directed to a hypothetical protein encoded by antisense p53 mRNA. Arpp protein is composed of 333 amino acids and contains four ankyrin-like repeat motifs in the middle portion of the protein, a PEST-like sequence and a lysine-rich sequence similar to a nuclear localization signal in the N-terminal region, and a proline-rich region containing consensus phosphorylation sites in the C-terminal region. Protein sequence analysis revealed that Arpp is homologous (52.7% identity) to Carp which is shown to be involved in the regulation of the transcription of the cardiac ventricular myosin light chain 2 gene. Arpp mRNA was found to be expressed in normal skeletal and cardiac muscle. Interestingly, Arpp expression was detectable in bilateral ventricles, but undetectable in bilateral atria and large vessels, suggesting that Arpp may play a specific function in cardiac ventricles as well as skeletal muscles.

Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory Light Chains of Myosin Affect Their Structure, Ca2+Binding, and Phosphorylation

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Congestive Heart Failure: Fifty Years of Progress

Congestive Heart Failure: Fifty Years of Progress

Restricted Expression of Cardiac Myosin Genes Reveals Regulated Aspects of Heart Tube Assembly in Zebrafish

The embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior–posterior (A–P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial–lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial–lateral pattern into the A–P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A–P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A–P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial–lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process.

Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2

A role for myosin phosphorylation in modulating normal cardiac function has long been suspected, and we hypothesized that changing the phosphorylation status of a cardiac myosin light chain might alter cardiac function in the whole animal. To test this directly, transgenic mice were created in which three potentially phosphorylatable serines in the ventricular isoform of the regulatory myosin light chain were mutated to alanines. Lines were obtained in which replacement of the endogenous species in the ventricle with the nonphosphorylatable, transgenically encoded protein was essentially complete. The mice show a spectrum of cardiovascular changes. As previously observed in skeletal muscle, Ca(2+) sensitivity of force development was dependent upon the phosphorylation status of the regulatory light chain. Structural abnormalities were detected by both gross histology and transmission electron microscopic analyses. Mature animals showed both atrial hypertrophy and dilatation. Echocardiographic analysis revealed that as a result of chamber enlargement, severe tricuspid valve insufficiency resulted in a detectable regurgitation jet. We conclude that regulated phosphorylation of the regulatory myosin light chains appears to play an important role in maintaining normal cardiac function over the lifetime of the animal.

Identification of a Novel Cardiac Lineage-Associated Protein(cCLP-1): A Candidate Regulator of Cardiogenesis

We describe the isolation and characterization of a cDNA clone, called cCLP-1, that is a candidate for the previously described early cardiac-specific transcription factor BBF-1. BBF-1 binds the MEF2 (or element B) binding site within the cardiac myosin light chain 2 (MLC2) gene promoter. We used the element B sequence as a probe to screen an expression library constructed from mRNA obtained from the presumptive heart-forming regions of stage 6 chicken embryos. This yielded the cCLP-1 cDNA clone. Gel-shift analysis of stage 6 embryonic chicken protein extracts suggests that a protein that is recognized by the anti-cCLP-1 antibody binds to the same element B binding site to which BBF-1 binds. cCLP-1 mRNA was detected early in chicken development, prior to cardiac fate assignment at stage 4. The expression pattern of cCLP-1, based on whole mountin situhybridization, coincides remarkably well with the established morphogenetic field of early heart formation. The nuclear localization of cCLP-1 is phosphorylation-dependent, suggesting that cCLP-1 may be a member of that class of transcription factors whose activity is regulated by cytoplasm to nucleus transport. Taken together, these data suggest that cCLP-1 may encode a novel transcription factor whose expression pattern is in agreement with that of the cardiogenic precursor cells of the early chicken embryo.

Pathogenesis of early cardiac myocyte damage after severe burns.

The importance of early cardiac myocyte damage during postburn trauma has been emphasized in recent years. However, its pathogenesis, prevention, and treatment have not been fully clarified. The aim of this study is to define its pathogenesis. Rats with 30% third-degree burns were used. Cardiac biochemical markers reflecting cardiac myocyte damage including troponin T, cardiac myosin light chain 1, creatinine kinase and its cardiac-specific isoenzyme compound, as well as inflammatory mediators such as tumor necrosis factor, endothelin/nitric oxide ratio, malondialdehyde, and superoxide dismutase, were determined. Cardiac biochemical markers reflecting cardiac myocyte damage, including troponin T, cardiac myosin light chain 1, cardiac-specific isoenzyme compound, were all significantly elevated between 3 hours and 24 hours after burn. Changes in tumor necrosis factor, endothelin/nitric oxide ratio, and malondialdehyde were similar to those of cardiac biochemical markers. In contrast, levels of superoxide dismutase declined markedly after burn. The findings of this study showed that considerable amounts of myocardial constructive protein degradation and release due to destruction of cardiac myocytes occurred early after severe burns. The inflammatory mediators released after burn injury may be involved in the pathogenesis of myocardial destruction.

Rhabdomyolysis in Patients with Hypoglycemic Coma. Three Cases Study.

We showed the three cases in rhabdomyolysis according to hypoglycemic coma. The measurements of creatine phosphokinase, creatine phosphokinase isozyme, myoglobin, and cardiac myosin light chain I are useful for diagnosing the rhabdomyolysis in patients with diabetes mellitus.

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

Serum levels of cardiac troponin I and troponin T in estimating myocardial infarct size soon after reperfusion

Cardiac troponin I (TnI) and troponin T (TnT) are highly specific myocardial markers. To determine whether their serum levels can be used to estimate myocardial infarct size soon after reperfusion. We measured the serum levels of TnI, TnT, and creatine kinase every 3 h, and the serum cardiac myosin light chain I (MLCI) every 24 h, in 42 patients with acute myocardial infarction in whom reperfusion therapy had successfully been performed. We calculated the severity of regional hypokinesis by analyzing the follow-up ventriculograms with the centerline method. The time from reperfusion to the peak level for TnI was 6.1 +/- 3.5 h, significantly shorter than those for creatine kinase (7.5 +/- 4.1 h) and MLCI (55 +/- 28 h). The time to peak level for TnT (6.8 +/- 4.0 h) differed significantly from that for MLCI but not from that for creatine kinase. There was a significant correlation between the peak levels of TnI and TnT (r = 0.86). The peak TnI and TnT levels were correlated well to the peak creatine kinase level (r = 0.67 and 0.69, respectively), total creatine kinase release (r = 0.66 and 0.66), and the peak MLCI level (r = 0.71 and 0.80). We observed excellent correlations between the peak levels of TnI and TnT, and regional hypokinesis (r = -0.84 and -0.85, respectively). These were comparable to the correlations between regional hypokinesis and the peak creatine kinase level (r = 0.75), total creatine kinase release (r = -0.72), and the peak MLCI level (r = -0.76). These results suggest that the peak serum levels of TnI and TnT in patients with successful reperfusion are accurate and early indices of infarct size.

Different actin affinities of human cardiac essential myosin light chain isoforms

The N terminus of myosin light chain 1 (MLC-1) of skeletal muscle bind to the C terminus of actin. We investigated whether the N termini of human cardiac MLC-1 isoforms likewise bind to actin. Furthermore, we investigated whether the N-terminal sequence 5–15 (P5–14) of MLC-1 of human atrium (ALC-1) and ventricle (VLC-1) bind with different affinities to actin. Affinity beads were produced by covalently coupling a synthetic peptide corresponding to the N-terminal sequence 4–14 of human VLC-1 to aminohexylagarose in order to bind G-actin. We found, that G-actin specifically binds to the affinity beads. Furthermore, preincubation of G-actin with P5-14 of both ALC-1 and VLC-1 decreased the amount of G-actin recovered from the affinity beads in a concentration-dependent manner. The half-maximal effective concentrations, however were significantly ( p<0.01) different being 0.32±0.02 μM and 0.71±0.02 μM for the VLC-1 and ALC-1 peptide, respectively. The appropriate scrambled peptides were without effect up to 3 μM. These results demonstrate the specific interaction between the N-terminal domains of human cardiac MLC-1 isoforms and actin and reveal different actin affinities of MLC-1 isoforms. Weak binding of ALC-1 to actin could explain the higher cycling kinetics of cross-bridges with ALC-1 compared to those with VLC-1.

Activation of CD8+T Lymphocytes in Insulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease directed against the insulin-secreting β cells of the islets of Langerhans of the pancreas. We have previously shown that in organ-specific autoimmune diseases, Graves’ disease (GD), and IDDM, the antigen that is specific for each of these disorders (i.e., TSH receptor for GD, glutamic acid decarboxylase-65 (GAD65) for IDDM) does not activate the disease-specific CD8+cells as fully as CD8+cells from normal persons. In order to identify the specific antigen responsible for triggering or maintaining autoimmunity in patients afflicted with the disease, we have studied the effects of islet (β) cell-specific antigens GAD65, insulin, pancreatic antigen (P69), T cell epitope 69 (Tep69), and a milk-derived bovine serum albumin (BSA)–peptide–ABBOS (pre-BSA positions 157–169) on the activation of CD8+T lymphocytes in IDDM patients. We compared the patterns of T cell activation with those mediated by an irrelevant peptide antigen, P348 (amino-terminal region of human cardiac myosin light chain-1), and also tetanus toxoid. We also studied the responses of CD8+T lymphocytes to these IDDM-relevant and -irrelevant antigens in Hashimoto's thyroiditis patients (HT), rheumatoid arthritis patients (RA), and normal control subjects (N) to compare the pattern of responses in the other autoimmune diseases. Activation of lymphocytes was monitored by measuring the expression of the activation molecule–major histocompatibility complex class II antigen (HLA–DR) on the surfaces of CD8+T lymphocytes by flow cytometry. Peripheral blood mononuclear cells (PBMC) obtained from 14 patients with IDDM, 14 N, 14 with HT, and 13 with RA were cultured for 7 days in the presence or absence of antigens. The stimulation index (SI) of activation of the lymphocytes was determined. When the response of CD8+T lymphocytes of IDDM patients to each of the IDDM-relevant antigens was compared to that of the irrelevant antigen, only GAD65 and ABBOS showed a significantly reduced activation compared to P348 and tetanus toxoid. Other relevant antigens, insulin, P69, and Tep69, did not show any significant differences in their SI compared to those of the irrelevant antigens. In the N, HT, and RA groups, there was no significant difference in the SI of the responses of CD8+cells to any of the relevant antigens compared to that of the irrelevant antigens. Moreover, CD8+T lymphocytes of IDDM patients showed a significantly lower activation by GAD65 than those from N, HT, and RA. In conclusion, our data suggest that CD8+T lymphocytes of IDDM patients but not those from N, HT, and RA groups have specifically reduced potential for activation in response to GAD65 but not to insulin, P69, and Tep69, whereas ABBOS exerts a less well-defined reductive effect on the activation of CD8+lymphocytes of IDDM patients. Since CD8+cells have been shown to contain suppressor activity, our data support the notion that a disease-specific defect in GAD65 autoantigenic induction of suppressor T lymphocytes may be important in the pathogenesis of IDDM.

Expression from cardiomyocyte-specific promoter after adenovirus-mediated gene transfer in vitro and in vivo

Adenoviruses are very attractive vectors for gene transfer into the cardiac muscle; however, their promiscuous tissue tropism, leading to an ectopic expression of the transgene, is a considerable practical limitation. To restrict expression of a reporter gene in cultured cardiomyocytes and in the heart of the rat, we have constructed a recombinant adenovirus (Ad-MLC2βgal) containing the β-galactosidase gene under the control of the rat ventricle-specific cardiac myosin light chain 2 (MLC-2v) promoter. We show in this work that the MLC-2v promoter inside the adenoviral genome retains its cardiac specificity in vitro in cultured cardiomyocytes as well as in vivo in the animal heart. Northern blot studies after Ad-MLC2βgal infection show significant transcription only in cells derived from the cardiac muscle and not from the skeletal muscle. Quantitative analysis of the β-galactosidase activity in a number of cell lines also confirms this result. The level of β-galactosidase expression in rat neonatal cardiomyocytes infected with Ad-MLC2βgal is 8% of that found when primary cells are infected with Ad-RSVβgal (containing a β-galactosidase gene under the control of the Rous sarcoma virus promoter). The cardiomyocytes-specific expression is also found after injection of Ad-MLC2βgal directly into the rat myocardium, although the viral genome can be detected by polymerase chain reaction (PCR) in other tissues. Lack of expression after direct injection into liver and skeletal muscle confirms these results. The use of a tissue-specific promoter is a first step to restrict transgene expression to a particular cell type of the targeted tissue.

Advantages of backscatter electron imaging scanning electron microscopy for intracellular localization of cardiac analytes by gold conjugated antibody.

Myoglobin and myosin light chain 1 (MLC1) are intracellular human cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.

Transcriptional regulation of the cardiac myosin light chain 2 gene under normal and stress conditions

Transcriptional regulation of the cardiac myosin light chain 2 gene under normal and stress conditions

Activation of T lymphocyte subsets by synthetic TSH receptor peptides and recombinant glutamate decarboxylase in autoimmune thyroid disease and insulin-dependent diabetes.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiac Myocyte Differentiation Induced by 3,5,3′-Triiodo-L-Thyronine (T 3) in P19-Teratocarcinoma Cells Is Accompanied by Preferential Binding of RGG(T/A)CA Direct Repeats Spaced by Four Base Pairs in the DNA

When treated with T 3, P19 cells differentiate into ultrastructurally proven cardiac myocytes and express the cardiac ventricular specific marker ventricular myosin light chain 2V. This differentiation is irreversibly induced in culture during the first 48 hrs of exposure to T 3. We studied the binding of P19-indigenous transcription factors of the Steroid-Thyroid-Retinoic superfamily of nuclear receptors to oligonucleotide response elements bearing direct, inverted and palindromic repeats of the consensus sequence RGG(T/A)CA. Electrophoretic mobility shift assays showed a preference in T 3-treated P19 cells for binding RGG(T/A)CA "half sites" in direct repeat orientation separated by 4 base pairs. The specificity of binding was confirmed in competition experiments. This finding suggests that target genes bearing thyroid response elements spaced by 4 base pairs in their promoter regions play an important role in the cardiac differentiation induced by T 3 in P19 teratocarcinoma cells.