Patrycja M. Dubielecka

Inhibition of Histone Deacetylase-induced Myocardial Repair Is Mediated by c-kit in Infarcted Hearts

Background: Histone deacetylases (HDACs) mediate cardiac development and diseases. Results: HDAC inhibition promoted cardiac repairs and neovascularization, which were absent in KitW/KitW-v mice. Preconditioning of c-kit+ cardiac stem cells via HDAC inhibition increased myocardial regeneration. Conclusion: HDAC inhibition promotes myocardial repair through c-kit signaling. Significance: The study advances our knowledge of myocardial repair and development of a novel therapeutic strategy for heart diseases. Histone deacetylases (HDACs) play a critical role in the regulation of gene transcription, cardiac development, and diseases. The aim of this study was to test whether inhibition of HDACs induces myocardial repair and cardiac function restoration through c-kit signaling in mouse myocardial infarction models. Myocardial infarction in wild type Kit+/+ and KitW/KitW-v mice was created following thoracotomy by applying permanent ligation to the left anterior descending artery. The HDAC inhibitor, trichostatin A (TSA, 0.1 mg/kg), was intraperitoneally injected daily for a consecutive 8 weeks after myocardial infarction. 5-Bromo-2-deoxyuridine (BrdU, 50 mg/kg) was intraperitoneally delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, inhibition of HDACs in vivo resulted in an improvement in ventricular functional recovery and the prevention of myocardial remodeling in Kit+/+mice, which was eliminated in KitW/KitW-v mice. HDAC inhibition promoted cardiac repairs and neovascularization in the infarcted myocardium, which were absent in KitW/KitW-v mice. Re-introduction of TSA-treated wild type c-kit+ CSCs into KitW/KitW-v myocardial infarction heart restored myocardial functional improvement and cardiac repair. To further validate that HDAC inhibition stimulates c-kit+ cardiac stem cells (CSCs) to facilitate myocardial repair, GFP+ c-kit+ CSCs were preconditioned with TSA (50 nmol/liter) for 24 h and re-introduced into infarcted hearts for 2 weeks. Preconditioning of c-kit+ CSCs via HDAC inhibition with trichostatin A significantly increased c-kit+ CSC-derived myocytes and microvessels and enhanced functional recovery in myocardial infarction hearts in vivo. Our results provide evidence that HDAC inhibition promotes myocardial repair and prevents cardiac remodeling, which is dependent upon c-kit signaling.

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca2+, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.

Differential regulation of macropinocytosis by Abi1/Hssh3bp1 isoforms.

Background Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown. Principal Findings Here, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably. Conclusion Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.

Crk and ABI1: binary molecular switches that regulate abl tyrosine kinase and signaling to the cytoskeleton.

The nonreceptor tyrosine kinases Abl and Arg are among the most well-characterized tyrosine kinases in the human genome. The activation of Abl by N-terminal fusions with Bcr (Bcr-Abl) or Gag (v-Abl) is responsible for chronic myeloid leukemia or Ph+ acute lymphoblastic leukemia and mouse leukemia virus, respectively. In addition, aberrant Abl and Arg activation downstream of several oncogenic growth factor receptors contributes to the development and progression of a variety of human cancers, often associated with poor clinical outcome, drug resistance, and tumor invasion and metastasis. Abl activation can occur by a variety of mechanisms that include domain interactions involving structural remodeling of autoinhibited conformations as well as direct phosphorylation by upstream kinases and phosphatases. Constitutive activation of Abl plays a significant role in regulating the actin cytoskeleton by modulating cell adhesion, motility, and invadopodia. This review addresses the role of Abl and Arg in tumor progression with particular emphasis on the roles of Crk and Abi1 adapter proteins as distinct molecular switches for Abl transactivation. These insights, combined with new insights into the structure of these kinases, provide the rationale to envision that Crk and Abi1 fine-tune Abl regulation to control signaling to the cytoskeleton.

Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI-3 kinase in regulation of macropinocytosis in LNCaP cells.

MINT‐7908027: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lck (uniprotkb:P06239) by array technology (MI:0008)

Irisin plays a pivotal role to protect the heart against ischemia and reperfusion injury

Irisin, a newly identified hormone, is critical to modulating body metabolism, thermogenesis and reducing oxidative stresses. However, whether irisin protects the heart against myocardial ischemia and reperfusion (I/R) injury remains unknown. In this study, we determine the effect of irisin on myocardial I/R injury in the Langendorff perfused heart and cultured myocytes. Adult C57/BL6 mice were treated with irisin (100 mg/kg) or vehicle for 30 min to elicit preconditioning. The isolated hearts were subjected to 30 min ischemia followed by 30 min reperfusion. Left ventricular function was measured and infarction size were determined using by tetrazolium staining. Western blot was employed to determine myocardial SOD‐1, active‐caspase 3, annexin V, p38, and phospho‐p38. H9c2 cardiomyoblasts were exposed to hypoxia and reoxygenation for assessment of the effects of irisin on mitochondrial respiration and mitochondrial permeability transition pore (mPTP). Irisin treatment produced remarkable improvements in ventricular functional recovery, as evident by the increase in RPP and attenuation in LVEDP. As compared to the vehicle treatment, irisin resulted in a marked reduction of myocardial infarct size. Notably, irisin treatment increased SOD‐1 and p38 phosphorylation, but suppressed levels of active‐caspase 3, cleaved PARP, and annexin V. In cardiomyoblasts exposed to hypoxia/reoxygenation, irisin treatment significantly attenuated hypoxia/reoxygenation (H/R), as indicated by the reduction of lactate dehydrogenase (LDH) leakage and apoptotic cardiomyocytes. Furthermore, irisin treatments suppressed the opening of mPTP, mitochondrial swelling, and protected mitochondria function. Our results indicate that irisin serves as a novel approach to eliciting cardioprotection, which is associated with the improvement of mitochondrial function.

Lipid-binding role of βII-spectrin ankyrin-binding domain

It is known that erythroid and non‐erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially‐expressed recombinant peptides representing βII(brain)‐spectrins ankyrin‐binding domain and its truncated mutants showed lipid‐binding activity, although only those containing a full‐length amino terminal fragment showed high to moderate affinity towards phospholipid mono‐ and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on βI‐spectrins ankyrin‐binding domain [Hryniewicz‐Jankowska A, et al. Mapping of ankyrin‐sensitive, PE/PC mono‐ and bilayer binding site in erythroid beta‐spectrin. Biochem J 2004;382:677–85]. Moreover, we tested also the effect of transient transfection of living cells of several cell‐lines with vectors coding for GFP‐conjugates including βII and also βI full‐length ankyrin‐binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full‐length ankyrin‐binding domain of βII and βI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin‐binding domain truncated at their N‐terminal region. Our results therefore indicate the importance of N‐terminal region for lipid‐binding activity of the β‐spectrin ankyrin‐binding domain and its substantial role in maintaining the spectrin‐based skeleton distribution.

Molecular targeting in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogenous disease associated with distinct genetic and molecular abnormalities. Somatic mutations result in dysregulation of intracellular signaling pathways, epigenetics, and apoptosis of the leukemia cells. Understanding the basis for the dysregulated processes provides the platform for the design of novel targeted therapy for AML patients. The effort to devise new targeted therapy has been helped by recent advances in methods for high-throughput genomic screening and the availability of computer-assisted techniques for the design of novel agents that are predicted to specifically inhibit the mutant molecules involved in these intracellular events. In this review, we will provide the scientific basis for targeting the dysregulated molecular mechanisms and discuss the agents currently being investigated, alone or in combination with chemotherapy, for treating patients with AML. Successes in molecular targeting will ultimately change the treatment paradigm for the disease.

The emerging role of protein kinase Cθ in cytoskeletal signaling.

Cytoskeletal rearrangements often occur as the result of transduction of signals from the extracellular environment. Efficient awakening of this powerful machinery requires multiple activation and deactivation steps, which usually involve phosphorylation or dephosphorylation of different signaling units by kinases and phosphatases, respectively. In this review, we discuss the signaling characteristics of one of the nPKC isoforms, PKCθ, focusing on PKCθ‐mediated signal transduction to cytoskeletal elements, which results in cellular rearrangements critical for cell type‐specific responses to stimuli. PKCθ is the major PKC isoform present in hematopoietic and skeletal muscle cells. PKCθ plays roles in T cell signaling through the IS, survival responses in adult T cells, and T cell FasL‐mediated apoptosis, all of which involve cytoskeletal rearrangements and relocation of this enzyme. PKCθ has been linked to the regulation of cell migration, lymphoid cell motility, and insulin signaling and resistance in skeletal muscle cells. Additional roles were suggested for PKCθ in mitosis and cell‐cycle regulation. Comprehensive understanding of cytoskeletal regulation and the cellular “modus operandi” of PKCθ holds promise for improving current therapeutic applications aimed at autoimmune diseases.