Aminoglycoside Phosphotransferase Research Articles

Backbone resonance assignments of a promiscuous aminoglycoside antibiotic resistance enzyme; the aminoglycoside phosphotransferase(3′)-IIIa

The aminoglycoside phosphotransferase(3')-IIIa (APH) is a promiscuous enzyme and renders a large number of structurally diverse aminoglycoside antibiotics useless against infectious bacteria. A remarkable property of this approximately 31 kDa enzyme is in its unusual dynamic behavior in solution; the apo-form of the enzyme exchanges all of its backbone amide protons within 15 h of exposure to D ( 2 ) O while aminoglycoside-bound forms retain approximately 40% of the amide protons even after >90 h of exposure. Moreover, the number of observable peaks and their dispersion in HSQC spectra varies with each aminoglycoside, rendering the resonance assignments very challenging. Therefore, the binary APH-tobramycin complex, which shows the largest number of well-resolved peaks, was used for the backbone resonance assignments (Calpha, C, N, H, and some Cbeta) of this protein (BMRB-16337).

NMR Detected Hydrogen−Deuterium Exchange Reveals Differential Dynamics of Antibiotic- and Nucleotide-Bound Aminoglycoside Phosphotransferase 3′-IIIa

In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3'-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both gram negative and gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycoside-modifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D(2)O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and approximately 30% of the amides remain unexchanged up to 95 h after exposure to D(2)O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby beta-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.

Deciphering interactions of the aminoglycoside phosphotransferase(3′)‐IIIa with its ligands

Aminoglycoside phosphotransferase(3')-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme-substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal-nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2'-NH(2) become better substrates (higher V(max)) than those with 2'-OH.

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

Alternate protein splicing mechanisms: A directed evolution approach

Inteins are intervening polypeptides that are able to direct their excision from the flanking polypeptides, called exteins, as well as facilitate the ligation of the exteins. This process, called protein splicing, produces the functional protein. We want to determine if inteins that splice by the standard mechanism also can splice via alternate mechanisms. First, we have inactivated splicing of the RecA intein from M. tuberculosis by mutating the N‐terminal Cys to Ala. Using saturation mutagenesis and error prone PCR, we generated random mutants that we subjected to a genetic screen to select for complementary mutations that may allow the modified intein to facilitate splicing by bypassing the first step. To select for complementary mutants, we used E. coli harboring a plasmid with the kanamycin resistance gene, aminoglycoside phosphotransferase, interrupted by the RecA intein, such that bacterial growth should depend on efficient splicing. We have been able to mutate the RecA intein and select inteins that allow for growth on kanamycin. However, these inteins do not promote splicing in our biochemical assay, which suggests that the intein may allow for a functional aminoglycoside phosphotransferase in the absence of splicing. This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.

Hydrogen‐Deuterium Exchange Reveals Antibiotic Resistance Enzyme Aminoglycoside Phosphotransferase 3′‐IIIa as Intrinsically Unstructured Protein

Hydrogen deuterium (H/D) exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3′‐IIIa (APH), which confer resistance to aminoglycoside antibiotics. This work represents the first characterization of dynamic properties of an aminoglycoside modifying enzyme. H/D exchange experiments performed with apo, binary and ternary complexes of APH with kanamycin A, neomycin B, and metal‐nucleotide showed that regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 hours of exposure to D2O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange and ~35% of the amides remain unexchanged up to 90 hours after exposure to D2O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to enzyme‐aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby β‐sheet thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide molecular basis for broad substrate selectivity of APH.

Search for Inhibitors of Bacterial and Human Protein Kinases among Derivatives of Diazepines[1,4] Annelated with Maleimide and Indole Cycles

Aminomethylation of 9b,10-dihydro-1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino-[1,7-a]indole-1,3(2H)-diones or 1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-diones resulted in dialkylaminomethyl derivatives. Alkylation of the nitrogen atom of maleimide moiety of polyannelated diazepines with 1,3-dibromopropane and subsequent reaction with thiourea or its N-alkyl derivatives gave isothiourea-carrying compounds. The compounds containing isothiourea moiety were active against individual human serine/threonine and tyrosine kinases at low micromolar concentrations. Dialkylaminomethyl derivatives of diazepines sensitized Streptomyces lividans with overexpressed aminoglycoside phosphotransferase type VIII (aphVIII) to kanamycin by inhibiting serine/threonine kinase(s) mediated aphVIII phosphorylation.

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca2+ ions. The inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca2+ -dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca2+/calmodulin and Ca2+/phospholipid-dependent STPK reduced the Ca2+ -induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.

Chaperone-Assisted Crystallography with DARPins

The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein--a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes.

Actinomycete integrative and conjugative pMEA-like elements of Amycolatopsis and Saccharopolyspora decoded

Actinomycete integrative and conjugative elements (AICEs) are present in diverse genera of the actinomycetes, the most important bacterial producers of bioactive secondary metabolites. Comparison of pMEA100 of Amycolatopsis mediterranei, pMEA300 of Amycolatopsis methanolica and pSE211 of Saccharopolyspora erythraea, and other AICEs, revealed a highly conserved structural organisation, consisting of four functional modules (replication, excision/integration, regulation, and conjugative transfer). Features conserved in all elements, or specific for a single element, are discussed and analysed. This study also revealed two novel putative AICEs (named pSE222 and pSE102) in the Sac. erythraea genome, related to the previously described pSE211 and pSE101 elements. Interestingly, pSE102 encodes a putative aminoglycoside phosphotransferase which may confer antibiotic resistance to the host. Furthermore, two of the six pSAM2-like insertions in the Streptomyces coelicolor genome described by Bentley et al. [Bentley, S.D., Chater, K.F., Cerdeno-Tarraga, A.M., et al., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147] could be functional AICEs. Homologues of various AICE proteins were found in other actinomycetes, in Frankia species and in the obligate marine genus Salinispora and may be part of novel AICEs as well. The data presented provide a better understanding of the origin and evolution of these elements, and their functional properties. Several AICEs are able to mobilise chromosomal markers, suggesting that they play an important role in horizontal gene transfer and spread of antibiotic resistance, but also in evolution of genome plasticity.

Aminoglycoside 2″-Phosphotransferase Type IIIa from Enterococcus

Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2''-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2''-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2''-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.

Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2''-phosphotransferase-Ic [APH(2'')-Ic] from Enterococcus gallinarum.

Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2''-phosphotransferase-Ic [APH(2'')-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2'')-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl(2), 0.1 M Tris-HCl pH 8.5 and 1 mM Mg(2)GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 A, beta = 108.8 degrees. X-ray diffraction data were collected to approximately 2.15 A resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

Isolation of antibiotic resistance bacterial strains from Eastern Siberia permafrost sediments

A collection of bacterial antibiotic resistance strains isolated from arctic permafrost subsoil sediments of various age and genesis was created. The collection included approximately 100 strains of Gram-positive (Firmicutes, Arthrobacter) and Gram-negative bacteria (Bacteroidetes, gamma-Proteobacteria, and alpha-Proteobacteria) resistant to aminoglycoside antibiotics (gentamycin, kanamycin, and streptomycin), chloramphenicol and tetracycline. Antibiotic resistance spectra were shown to differ in Gram-positive and Gram-negative bacteria. Multidrug resistance strains were found for the first time in ancient bacteria. In studies of the molecular nature of determinants for streptomycin resistance, determinants of the two types were detected: strA-strB genes coding for aminoglycoside phosphotransferases and genes aadA encoding aminoglycoside adenylyltransferases. These genes proved to be highly homologous to those of contemporary bacteria.

Detection of Specific Solvent Rearrangement Regions of an Enzyme: NMR and ITC Studies with Aminoglycoside Phosphotransferase(3‘)-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.

A New Helicobacter pylori Vacuolating Cytotoxin Determinant, the Intermediate Region, Is Associated With Gastric Cancer

Helicobacter pylori is the main cause of peptic ulceration and gastric adenocarcinoma. The vacuolating cytotoxin gene, vacA, is a major determinant of virulence. Two naturally polymorphic sites in vacA, the signal region and midregion, are well-characterized determinants of toxicity and markers of pathogenesis. The aim of this study was to characterize a new vacA polymorphic site, the intermediate (i) region. The vacA i-region was identified and characterized by constructing isogenic vacA exchange mutants and determining their vacuolating activity on HeLa, AGS, and RK13 cell lines. The vacA i-region types of H pylori isolates from patients undergoing routine endoscopy were determined by nucleotide sequencing and allele-specific polymerase chain reaction. Two i-region types were identified, i1 and i2, and both were common among 42 Western clinical isolates. Interestingly, only naturally occurring s1/m2 strains varied in i-type; s1/m1 and s2/m2 strains were exclusively i1 and i2, respectively. Vacuolation assays showed that i-type determined vacuolating activity among these s1/m2 strains, and exchange mutagenesis confirmed that the i-region itself was directly responsible. Using a simple i-region polymerase chain reaction-based typing system, it was shown for 73 Iranian patients that i1-type strains were strongly associated with gastric adenocarcinoma (P < 10(-3)). Finally, logistic regression analysis showed this association to be independent of, and larger than, associations of vacA s- or m-type or cag status with gastric adenocarcinoma. Together these data show that the vacA i-region is an important determinant of H pylori toxicity and the best independent marker of VacA-associated pathogenicity.

Photoaffinity labeling of Aminoglycoside Phosphotransferase 3′‐II confirms the involvement of conserved lysine in ATP binding

Photoaffinity labeling of Aminoglycoside Phosphotransferase 3′‐II confirms the involvement of conserved lysine in ATP binding

Molecular Determinants of Antibiotic Recognition and Resistance by Aminoglycoside Phosphotransferase (3′)-IIIa: A Calorimetric and Mutational Analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3′)-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with 15N NMR-derived pKa and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2′-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3′)-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3′)-IIIa-induced resistance.

Aph(3′)-IIc, an Aminoglycoside Resistance Determinant from Stenotrophomonas maltophilia

We report the characterization of an intrinsic, chromosomally carried aph(3')-IIc gene from Stenotrophomonas maltophilia clinical isolate K279a, encoding an aminoglycoside phosphotransferase enzyme that significantly increases MICs of kanamycin, neomycin, butirosin, and paromomycin when expressed in Escherichia coli. Disruption of aph(3')-IIc in K279a results in decreased MICs of these drugs.

Dissection of Aminoglycoside−Enzyme Interactions: A Calorimetric and NMR Study of Neomycin B Binding to the Aminoglycoside Phosphotransferase(3‘)-IIIa

In this work, for the first time, we report pKa values of the amino functions in a target-bound aminoglycoside antibiotic, which permitted dissection of the thermodynamic properties of an enzyme-aminoglycoside complex. Uniformly enriched 15N-neomycin was isolated from cultures of Streptomyces fradiae and used to study its binding to the aminoglycoside phosphotransferase(3')-IIIa (APH) by 15N NMR spectroscopy. 15N NMR studies showed that binding of neomycin to APH causes upshifts of approximately 1 pKa unit for the amines N2' and N2' '' while N6' experienced a 0.3 pKa unit shift. The pKa of N6' '' remained unaltered, and resonances of N1 and N3 showed significant broadening upon binding to the enzyme. The binding-linked protonation and pH dependence of the association constant (Kb) for the enzyme-aminoglycoside complex was determined by isothermal titration calorimetry. The enthalpy of binding became more favorable (negative) with increasing pH. At high pH, binding-linked protonation was attributable mostly to the amino functions of neomycin; however, at neutral pH, functional groups of the enzyme, possibly remote from the active site, also underwent protonation/deprotonation upon formation of the binary enzyme-neomycin complex. The Kb for the enzyme-neomycin complex showed a complicated dependence on pH, indicating that multiple interactions may affect the affinity of the ligand to the enzyme and altered conditions, such as pH, may favor one or another. This work highlights the importance of determining thermodynamic parameters of aminoglycoside-target interactions under different conditions before making attributions to specific sites and their effects on these global parameters.

Cardiomyocyte production in mass suspension culture: Embryonic stem cells as a source for great amounts of functional cardiomyocytes

Transplantation of cardiomyocytes derived from embryonic stem (ES) cells into damaged or non-functional areas of the myocardium might be a valuable approach to treat heart failure, provided that they can be generated in sufficient quantity and with sufficient purity. Cultivation of ES cells in a standard stirred tank bioreactor makes it possible to generate large numbers of therapeutically useful cells such as cardiomyocytes. We have used this technique to expand genetically engineered ES cells transfected with a fusion gene consisting of the alpha-cardiac myosin heavy chain (MyHc) promotor driving the aminoglycoside phosphotransferase gene (neomycin resistance). Addition of G418 to the growth medium allowed an efficient enrichment of cardiomyocytes. We developed a draw-and-fill process in a controlled stirred tank reactor at the 2L scale to facilitate long-term cultivation under stable, reproducible conditions. Throughout the culture period cell viability and EB numbers were measured monitored by flow cytometry, immunohistochemistry and fluorescence microscopy of cardiac specific antigens. About 9e8 cardiomyocytes in 2e5 cardiac bodies were generated. The purity of the population was about 99% measured by MF20 immunohistochemistry and fluorescence microscopy. To analyze the ability of ES-cell derived cardiomyocytes to reconstitute damaged heart tissue cardiac bodies were transplanted into female ICR-mice 7 days after myocardial infarction induced by LAD ligation. Both echocardiographic follow-up and histological analysis 28d after transplantation showed an improved left ventricular function of hearts that received a transplant in comparison to sham-treated mice. We here demonstrate that it is feasible to generate functional cardiomyocytes derived from embryonic stem cells in a bioreactor at a large-scale that retain their functionally in vivo and improve cardiac functions of infarcted hearts. With only one bioreactor process it is possible to provide sufficient cardiomyocytes for a cell based therapy.