[Interventional, intramyocardial stem cell therapy in ischemic cardiomyopathy: update 2010]. / Aktuelle Datenlage zur interventionellen, intramyokardialen Stammzelltherapie bei ischämischer Kardiomyopathie.

BACKGROUND: The intracoronary application of autologous bone marrow cells has proven hitherto to be safe but not sufficiently effective in patients with ischemic cardiomyopathy. The interventional application of cells injected directly into the myocardium represents one possible approach to improve effectiveness. TECHNIQUES: The NOGA method is based on the CARTO technique, which has been evaluated extensively for safety and feasibility in patients with heart failure. In a first step, an electrically and anatomically exact map of the left ventricle is obtained. Guided by this three-dimensional map direct injection of the cells into the ischemic area can be easily performed. CLINICAL STUDIES: Since its introduction in 2002 many studies have proven the safety, feasibility and effectiveness of NOGA-guided regenerative therapy to the left ventricle. While several studies also suggest effectiveness regarding various parameters of left ventricular function, no larger multicenter study is available to date. Such studies with also clinical endpoints are currently ongoing. CONCLUSION: The currently available data support, but do not yet prove, the hypothesis that intramyocardial stem cell therapy using NOGA-guided injection into the myocardium is safe and feasible in both acute and chronic ischemic cardiomyopathy. Ongoing trials will reveal whether this approach will become the standard form for applying cell therapy to the heart.

Percutaneous intramyocardial stem cell injection in patients with acute myocardial infarction: first-in-man study.

BACKGROUND: Clinical studies on intracoronary stem cell infusion in patients with acute myocardial infarction (AMI) have shown promising results for left ventricular ejection fraction (LVEF). However, preclinical studies have shown that intramyocardial cell injection is better than the intracoronary approach. OBJECTIVE: To test safety and feasibility of intramyocardial cell injection and left ventricular electromechanical mapping (EMM) early after AMI. DESIGN: On day 10.5 (5) (mean (SD)) after AMI and percutaneous coronary intervention with stent implantation (culprit lesion: 15 LAD, 3 circumflex and 2 right coronary arteries) 20 patients (mean (SD) 60.4 (11.4) years) received bone marrow derived mononuclear cells in the low-voltage area using EMM-guided percutaneous intramyocardial injection. EMM and coronary angiography were performed in 15 patients at 6-months' follow-up. Echocardiography, recording of laboratory data and clinical assessment (6-month and 12-month follow-up) were carried out in all 20 patients. RESULTS: None of the patients showed periprocedural complications. Three patients received an implantable cardioverter-defibrillator for primary prevention of sudden cardiac death and 6 (30%) patients showed in-stent restenosis. One patient underwent bypass surgery owing to chronic stent occlusion after 6 months. 2.0 (0.6)x10(8) cells, including 1.0 (0.3)x10(6) CD45(dim)/CD34(hi) stem cells, were injected in each patient. EMM showed a mean (SD) improvement from a baseline unipolar voltage of 45.5 (14.3)% to 59.3 (19.8)% of normal voltage (p = 0.002) and reduction of the low-voltage area from 28.7 (12.1)% to 20.3 (13.5)%; (p = 0.016). During the 12-month follow-up, the left ventricular ejection fraction (LVEF) improved from 40.8 (6.9)% to 47.1 (10.6)%; (p = 0.037). CONCLUSION: Left ventricular EMM and percutaneous intramyocardial cell injection in patients with AMI was shown to be a safe procedure. It is associated with improved LVEF and electromechanical parameters after 12-months' follow-up. TRIAL REGISTRATION NUMBER: Eudra-CT-No 2005-003629-19.

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization.

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.

A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus.

Bisphosphorylation of two adjacently located serine residues in the heart-specific N-terminus of the cTnl subunit reduces calcium affinity of the cTnC subunit. An interaction of the phosphorylation region of cTnI with acidic residues of another cTn subunit has been proposed formerly based on 31P nuclear magnetic resonance (NMR) data. A possible candidate is cTnC. Thus, an interaction model of cTnC with the bisphosphorylated cTnI N-terminus has been built using a homology model of hcTnC based on the crystal structure of tusTnC and the structure of the phosphorylation region of cTnI determined by 2D NMR. By computational search, five cluster of acidic residues of cTnC might interact with the cTnI phosphorylation region. Three sites could be excluded by 31P-NMR experiments. The two remaining sites are located in the N-terminal helix of cTnC and between calcium binding sites III and IV. Reorientation of the arginine and phosphoserine sidechains within the phosphorylation region as proposed by refined docking could explain the formerly measured changes in pKaapp values. Thus, local pKa changes might lead to the reduction of calcium affinity observed upon cTnI bisphosphorylation.

Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I.

Four phosphorylation degrees of cardiac troponin I (cTnI) have been characterized, namely, a dephospho, a bisphospho, and two monophospho states. Here we describe for the first time a role of the monophosphorylated forms. We have investigated the interaction between the cardiac troponin subunits dependent on the phosphorylation state of cTnI by surface plasmon resonance (SPR) spectroscopy. The monophosphorylated forms were generated by mutating each of the two serine residues, located in human cTnI at positions 22 and 23, to alanine. Association and dissociation rate constants of binary (cTnI-cTnT and cTnI-cTnC) and ternary (cTnI/cTnC complex-cTnT) complexes were determined. Mono- and consecutive bisphosphorylation of cTnI gradually reduces the affinity to cTnC and cTnT by lowering the association rate constants; the dissociation rate constants remain unchanged. Phosphorylation also affects formation of the ternary complexes; however, in this instance, association rate constants are constant, and dissociation rate constants are enhanced. A model of cardiac troponin is presented describing an induction of distinct conformational changes by mono- and bisphosphorylation of cTnI.

Microanalysis and distribution of cardiac troponin I phospho species in heart areas.

Sequential phosphorylation and dephosphorylation of cTnI by the cAMP dependent protein kinase and by protein phosphatase 2A, respectively, produce the non-, mono- and bisphosphorylated species (Jaquet et al., 1995, Eur. J. Biochem. 231, 486-490). The aim of this study was to determine these forms even in small tissue samples, e.g. in biopsy probes of approximately 30 mg which would allow to define the phosphorylation state of cTnI in heart areas. In order to do so a micro isolation procedure for cTnI had to be established. cTnI is extracted from small bovine, rabbit and human heart tissue samples (30-100 mg) under special conditions avoiding dephosphorylation and is isolated by affinity chromatography on cTnC Sepharose. All three species, the bis-, mono- and dephospho cTnI, are precipitated quantitatively by acetone, then they are separated by non-equilibrium isoelectric focusing and quantified by scanning densitometry. The method presented here allows to quantify the three cTnI species reproducibly. No other phosphorylated species are detected. Truncated cTnI forms of each phospho species are found in human biopsy samples due to removal of a approximately 36 amino acid peptide from the C-terminus. In bovine, human and rabbit heart the pattern of the three cTnI phospho species is characteristic for left and right atrium, left and right ventricle and septum.

Functional intactness of stimulatory and inhibitory autocrine loops in human renal carcinoma cell lines of the clear cell type.

PURPOSE: The aim of the present study was to analyze the contribution of different stimulatory and inhibitory growth factors to the deregulated proliferation of human RCCs. MATERIALS AND METHODS: The expression of different growth factors and their corresponding receptors were analyzed by Northern blot, FACS, ELISA and immunocytochemistry in 13 permanent human RCC cell lines of the clear cell type. Moreover, the functional intactness of growth factor-related signal transduction pathways was investigated. RESULTS: All RCC cell lines expressed EGF-receptor mRNA and protein and 10 cell lines secreted TGF-alpha. Exogeneously added TGF-alpha resulted in a significant (p < 0.05) stimulation of growth in 6 RCC cell lines and a significant (p < 0.05) inhibition of proliferation in 3 cell lines. PDGF B and the corresponding type beta receptor were expressed in a single cell line. mRNA expression of PDGF A and PDGF-alpha-receptor as well as IGF-1 and its receptor could not be detected in any cell line. Eleven RCC cell lines expressed TGF-beta 1 mRNA and in all cell lines TGF-beta 1 secretion into the supernatant could be demonstrated. Whereas all cell lines exhibited TGF-beta type II-receptor mRNA, type I-receptor mRNA could be detected only in 3 cell lines. TGF-beta type III-receptor was observed in 1 cell line. Exogeneously added TGF-beta1 resulted in a significant (p < 0.05) inhibition of proliferation in 7 RCC cell lines. CONCLUSION: Clear cell RCCs exhibit a complex and heterogeneous expression pattern for various growth factors and their receptors. Growth factor secretion and intact signal transduction pathways in most clear cell RCCs facilitate an intricate modulation of RCC growth by autocrine and paracrine interactions between tumor cells and host tissue.

Acquisition of TGF-beta 1 resistance: an important progression factor in human renal cell carcinoma.

The balance of growth regulation in tumors can be profoundly disturbed by defects in the transforming growth factor-beta 1 (TGF-beta 1) system. Thus far, investigations into the secretion of TGF-beta 1, the expression of its type I, II, and III receptors, as well as the functional intactness of the TGF-beta 1 signal transduction pathways in human renal cell carcinomas (RCC) have been lacking. The objective of the present study, therefore, was to elucidate the role of the TGF-beta 1 system in RCC. We were able to determine the status of this system in 20 primary RCC and in 30 newly established human RCC cell lines of all major histologic types. All primary RCC showed expression of TGF-beta 1 and its type I and II receptors by immunohistochemistry, irrespective of histologic type. In vitro, all RCC cell lines secreted TGF-beta 1 protein as a biologically inactive complex, which cannot interact with cell-surface receptors. Type I ALK-5-receptor mRNA and protein were detected in 29 RCC cell lines, whereas type I ALK-2-receptor mRNA was found in all cell lines. Type II-receptor mRNA and protein could be demonstrated in all cell lines analyzed, and type III-receptor mRNA was observed in five RCC cell lines. Exogenously added, biologically active TGF-beta 1 (1 ng/ml) resulted in significant (p < 0.05) inhibition of proliferation in 14 of 30 RCC cell lines. Sixteen of thirty RCC cell lines, however, proved to be TGF-beta 1-resistant. This resistance could not be explained by mutations in two "hot spot" regions of the type II-receptor gene (bp 622 to 795 and bp 1868 to 2019), as was demonstrated by DNA sequencing in the TGF-beta 1-resistant RCC cell lines. In conclusion, our observations are the first to provide evidence of an "escape" from the negative growth control of TGF-beta 1 by a significant proportion of RCC, suggesting that the acquisition of TGF-beta 1 resistance is an important progression factor for human RCC.

Bisphosphorylation of cardiac troponin I modulates the Ca(2+)-dependent binding of myosin subfragment S1 to reconstituted thin filaments.

We have reconstituted thin filaments comprising pyrene-labelled actin (pyr-actin), tropomyosin (Tm) and cardiac troponin (cTn). cTn was isolated in two defined phosphorylation states; completely dephosphorylated on all subunits and with only the cTnI subunit bisphosphorylated. The thin filament was saturated with cTn at a pyr-actin/Tm/cTn ratio of 7:1:1. The calcium-dependent binding of S1 to thin filaments was measured in a stopped-flow spectrophotometer and the dependence of the observed rate constant on [Ca2+] fitted to the Hill equation. The only significant difference between the two phosphorylation states of the filaments was a 0.36 decrease in the pCa50 on bisphosphorylation.

Pattern formation on cardiac troponin I by consecutive phosphorylation and dephosphorylation.

Two serine residues located adjacently in the heart-specific N-terminus of cardiac troponin I can be phosphorylated in vivo. Both residues are sequentially phosphorylated and dephosphorylated by cAMP-dependent protein kinase (PKA) and protein phosphatase 2A (PP2A). The concentration changes of the different troponin I species have been determined separately for the phosphorylation and dephosphorylation reaction and approximated by time courses predicted by a reaction model. Dependent on the concentration ratio of active protein kinase/protein phosphatase, four different troponin I species can be generated; one nonphosphorylated, two monophosphorylated and one bisphosphorylated. This pattern generation will be observed in proteins phosphorylated and dephosphorylated by a single protein kinase and phosphatase on more than one site and is a new principle inherent in signal cascades.

A site phosphorylated in bovine cardiac troponin T by cardiac CaM kinase II.

Ca2+/Calmodulin-dependent protein kinase II isolated from heart phosphorylates bovine cardiac troponin T present in the holotroponin complex. Thr-190 has been determined as the main phosphorylation site.

Characterization of the cardiac troponin I phosphorylation domain by 31P nuclear magnetic resonance spectroscopy.

Cardiac holotroponin can be phosphorylated at serine 23 and/or 24 in the heart-specific region of bovine troponin I. When isolated freshly it is composed of a mixture of non-, two mono-, and bisphosphorylated species. At neutral pH the monophosphorylated form carrying phosphate at serine 24 yields a resonance signal at 4.6 ppm and that carrying phosphate at serine 23 at 4.4 ppm; the two phosphate groups of the bisphosphorylated form yield only one 31P-NMR signal at 4.2 ppm. From the chemical shift dependence on pH, pKa values have been estimated to be 5.3 and 5.6 for the phosphate groups at serine 24 and serine 23, respectively. Both phosphates of the bisphosphorylated form exhibit very similar pKa values of approximately 5.8. Separation of bisphosphotroponin I from the complex results in a downfield shift and the appearance of two 31P-NMR signals at positions comparable to those of the two monophospho forms. Complex formation of cardiac troponin I with C or T does not alter the spectrum obtained with isolated troponin I; however, the original troponin spectrum is restored by reconstitution of the holocomplex from all three components T, I, and C. Two signals are also observed with a bisphosphorylated synthetic peptide [PVRRRS(P)S(P)ANYR] representing the phosphorylation domain. pKa values of about 5.3 and 5.6 have been determined for serine 7 (corresponding to serine 24 of troponin I) and serine 6 of the peptide (corresponding to serine 23 of troponin I).

Clonal analysis of agnogenic myeloid metaplasia.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.

DNA analysis to aid in the diagnosis of chronic myeloproliferative disorders.

Diagnosing chronic myeloproliferative disorders (CMPD) can be difficult because of overlap and possible transitions between the different conditions and their similarity to reactive myeloproliferations. DNA analysis was applied to improve differentiation of CMPDs. All subtypes of CMPD analyzed, including chronic myeloid leukemia, agnogenic myeloid metaplasia, polycythemia vera, and essential thrombocythemia, had in common that granulocytes and bone marrow cells were clonal in origin, as shown by X chromosome-linked DNA polymorphism in conjunction with methylation patterns (n = 32). Reactive myeloproliferations, by contrast, showed polyclonal inactivation patterns. Clonality could not distinguish CMPD from cases of myelodysplastic syndrome because the latter (n = 7) also exhibited clonal hematopoiesis. Because of their clonal origin, peripheral granulocytes were used in all cases (n = 201) to detect bcr gene rearrangement. Despite possible morphologic overlap between different types of CMPD, bcr gene rearrangement was specific for chronic myeloid leukemia and could be applied to differentiate chronic myeloid leukemia from other CMPDs in cases of equivocal morphologic diagnosis. Chronic myeloproliferative disorders represent clonal hemopoietic diseases that probably have specific underlying genetic defects. Thus DNA analysis can aid substantially in the differential diagnosis of CMPD.

Ordered phosphorylation of a duplicated minimal recognition motif for cAMP-dependent protein kinase present in cardiac troponin I.

Cardiac troponin I contains two adjacent serines in sequence after three arginine residues thus making up a minimally duplicated recognition motif for cAMP-dependent protein kinase. In a synthetic peptide, PVRRRSSANY, the two serine residues are phosphorylated sequentially with the intermediate formation of a monophosphorylated species according to the following reaction sequence: Peptide k1----Peptide-P k2----Peptide-P2. The calculated rat constants are: k1 = 0.435.min-1 and k2 = 0.034.min-1. Sequence analyses of the monophosphopeptide and its tryptic fragments show that the predominant monophosphoform carries phosphate at the second serine.

Lineage-specific methylation of the c-fms gene in blood cells and macrophages.

DNA methylation belongs to the multilevel genetic control system regulating differentiation processes and gene expression. The extent to which DNA methylation contributes to the differentiation of hematopoietic cells is elusive. In the present study we investigated the methylation state of the c-fms/M-CSF receptor gene in normal human blood cells and tissue macrophages. The methylation pattern of the c-fms gene as detected by isoschizomeric restriction analysis with MspI/HpaII showed only slight interindividual variations in normal donors, whereas constant differences were found between granulocytes and monocytes from the same donor. The second intron of the c-fms gene contains several CpG loci which were found to be hypomethylated on both alleles in monocytes and tissue macrophages. By contrast, these positions were methylated in granulocytes and lymphocytes that did not express the c-fms gene. In comparison to monocytes alveolar and peritoneal macrophages revealed an enhanced demethylation. There were constant differences in c-fms gene methylation between alveolar and peritoneal macrophages with a higher degree of demethylation in alveolar macrophages. We conclude that c-fms gene demethylation is involved in the differentiation of monocytes and macrophages from immature precursors and that the demethylation of lineage-specific growth factor receptor genes might provide an important step in lineage commitment of hematopoietic cells.