Oleksandr Platoshyn

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.

Mechanisms of Hybrid Oligomer Formation in the Pathogenesis of Combined Alzheimer's and Parkinson's Diseases

Background Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimers disease (AD) and Parkinsons disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear. Methodology/Principal Findings In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels. Conclusion/Significance These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Significance Mutations in the RNA binding protein TDP-43 cause amyotrophic lateral sclerosis and frontotemporal dementia. Through expressing disease-causing mutants in mice and genome-wide RNA splicing analyses, mutant TDP-43 is shown to retain normal or enhanced activity for facilitating splicing of some RNA targets, but “loss-of-function” for others. These splicing changes, as well as age-dependent, mutant-dependent lower motor neuron disease, occur without loss of nuclear TDP-43 or accumulation of insoluble aggregates of TDP-43. Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43Q331K and TDP-43M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.

Dynamics of α-synuclein aggregation and inhibition of pore-like oligomer development by β-synuclein

Accumulation of α‐synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinsons disease and Lewy body dementia. In contrast, β‐synuclein (β‐syn), a close homologue, does not aggregate and reduces α‐synuclein (α‐syn)‐related pathology. Although considerable information is available about the conformation of α‐syn at the initial and end stages of fibrillation, less is known about the dynamic process of α‐syn conversion to oligomers and how interactions with antiaggregation chaperones such as β‐synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle‐derived structure of α‐syn showed that α‐syn homodimers can adopt nonpropagating (head‐to‐tail) and propagating (head‐to‐head) conformations. Propagating α‐syn dimers on the membrane incorporate additional α‐syn molecules, leading to the formation of pentamers and hexamers forming a ring‐like structure. In contrast, β‐syn dimers do not propagate and block the aggregation of α‐syn into ring‐like oligomers. Under in vitro cell‐free conditions, α‐syn aggregates formed ring‐like structures that were disrupted by β‐syn. Similarly, cells expressing α‐syn displayed increased ion current activity consistent with the formation of Zn2+‐sensitive nonselective cation channels. These results support the contention that in Parkinsons disease and Lewy body dementia, α‐syn oligomers on the membrane might form pore‐like structures, and that the beneficial effects of β‐synuclein might be related to its ability to block the formation of pore‐like structures.

Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice

Vascular endothelial cells (ECs) play a major role in regulating vascular tone and in revascularization. There is increasing evidence showing endothelial dysfunction in diabetes, although little is known about the contribution of connexins (Cxs) to vascular complications in the diabetic heart. This study was designed to investigate the role of Cxs in coronary endothelial dysfunction in diabetic mice. Coronary ECs isolated from diabetic mice exhibit lowered protein levels of Cx37 and Cx40 (but not Cx43) and a loss of gap junction intercellular communication (GJIC). Vasodilatation induced by the assumed contribution of EC-dependent hyperpolarization was significantly reduced in the diabetic coronary artery (CA). Cx40-specific inhibitory peptide (40)GAP27 strongly attenuated endothelium-dependent relaxation in diabetic CA at the concentration that does not affect the relaxation in control CA, suggesting that the total amount of Cx40 is lower in diabetic CA than in control CA. In diabetic mice, coronary capillary density was significantly decreased in vivo. In vitro, GJIC inhibitor attenuated the ability of EC capillary network formation. High-glucose treatment caused a decrease in Cx40 protein expression in ECs and impaired endothelial capillary network formation, which was restored by Cx40 overexpression. Furthermore, we found that the hyperglycemia-induced decrease in Cx40 was associated with inhibited protein expression of Sp1, a transcriptional factor that regulates Cx40 expression. These data suggest that downregulation of Cx40 protein expression and resultant inhibition of GJIC contribute to coronary vascular dysfunction in diabetes.

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation

IntroductionIntraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury.MethodsThree-month-old female Sprague–Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies.ResultsIntraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons.ConclusionsPeri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

Functional ion channels in human pulmonary artery smooth muscle cells: Voltage-dependent cation channels

The activity of voltage-gated ion channels is critical for the maintenance of cellular membrane potential and generation of action potentials. In turn, membrane potential regulates cellular ion homeostasis, triggering the opening and closing of ion channels in the plasma membrane and, thus, enabling ion transport across the membrane. Such transmembrane ion fluxes are important for excitation-contraction coupling in pulmonary artery smooth muscle cells (PASMC). Families of voltage-dependent cation channels known to be present in PASMC include voltage-gated K+ (Kv) channels, voltage-dependent Ca2+-activated K+ (Kca) channels, L- and T-type voltage-dependent Ca2+ channels, voltage-gated Na+ channels and voltage-gated proton channels. When cells are dialyzed with Ca2+-free K+-solutions, depolarization elicits four components of 4-aminopyridine (4-AP)-sensitive Kvcurrents based on the kinetics of current activation and inactivation. In cell-attached membrane patches, depolarization elicits a wide range of single-channel K+ currents, with conductances ranging between 6 and 290 pS. Macroscopic 4-AP-sensitive Kv currents and iberiotoxin-sensitive Kca currents are also observed. Transcripts of (a) two Na+ channel α-subunit genes (SCN5A and SCN6A), (b) six Ca2+ channel α-subunit genes (α1A, α1B, α1x, α1D, α1E and α1G) and many regulatory subunits (α2δ1, β1-4, and γ6), (c) 22 Kv channel α-subunit genes (Kv1.1 – Kv1.7, Kv1.10, Kv2.1, Kv3.1, Kv3.3, Kv3.4, Kv4.1, Kv4.2, Kv5.1, Kv 6.1-Kv6.3, Kv9.1, Kv9.3, Kv10.1 and Kv11.1) and three Kv channel β-subunit genes (Kvβ1-3) and (d) four Kca channel α-subunit genes (S/oα1 and SK2-SK4) and four Kca channel β-subunit genes (Kcaβ1-4) have been detected in PASMC. Tetrodotoxin-sensitive and rapidly inactivating Na+ currents have been recorded with properties similar to those in cardiac myocytes. In the presence of 20 mM external Ca2+, membrane depolarization from a holding potential of −100 mV elicits a rapidly inactivating T-type Ca2+ current, while depolarization from a holding potential of −70 mV elicits a slowly inactivating dihydropyridine-sensitive L-type Ca2+ current. This review will focus on describing the electrophysiological properties and molecular identities of these voltage-dependent cation channels in PASMC and their contribution to the regulation of pulmonary vascular function and its potential role in the pathogenesis of pulmonary vascular disease.

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes

ABSTRACT Vinculin (Vcl) links actin filaments to integrin- and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and &bgr;1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

Divergent effects of BMP-2 on gene expression in pulmonary artery smooth muscle cells from normal subjects and patients with idiopathic pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in pulmonary artery smooth muscle cells (PASMCs) from normal subjects. Dysfunction of BMP signaling due to mutations in and/or down-regulation of BMP receptors has been implicated in idiopathic pulmonary arterial hypertension (IPAH). The authors examined whether BMP differentially regulates gene expression in PASMCs from normal subjects and IPAH patients using the Affymetrix microarray analysis. BMP-2 treatment (200 nM for 24 hours) altered expression levels of 6206 genes in normal and IPAH PASMCs. Of these genes, 1063 were regulated oppositely by BMP-2: 523 genes were down-regulated by BMP-2 in normal PASMCs but up-regulated in IPAH PASMCs, whereas 540 genes were up-regulated by BMP-2 in normal PASMCs but down-regulated in IPAH PASMCs. The divergent effects of BMP-2 on gene expression profiles indicate that PASMCs may undergo significant phenotypic changes in IPAH patients during development of the disease. The transition of the antiproliferative effect of BMP-2 in normal PASMCs to its proliferative effect in IPAH patients is attributed potentially to its differential effect on expression patterns of various genes that are involved in cell proliferation and apoptosis. Among the 6206 BMP-2–sensitive genes, there are more than 1800 genes whose expression levels were negatively (correlation coefficient, r, <−0.9) or positively (with r >+ 0.9) correlated with the pulmonary arterial pressure. These results suggest that BMP-mediated gene regulation is significantly altered in PASMCs from IPAH patients and mRNA expression changes in BMP-regulated genes may be involved in the development of IPAH.

Identification of functional voltage-gated Na+ channels in cultured human pulmonary artery smooth muscle cells

Electrical excitability, which plays an important role in excitation–contraction coupling in the pulmonary vasculature, is regulated by transmembrane ion flux in pulmonary artery smooth muscle cells (PASMC). This study aimed to characterize the electrophysiological properties and molecular identities of voltage-gated Na+ channels in cultured human PASMC. We recorded tetrodotoxin (TTX) sensitive and rapidly inactivating Na+ currents with properties similar to those described in cardiac myocytes. Using RT-PCR, we detected transcripts of seven Na+ channel α genes (SCN2A, 3A, 4A, 7A, 8A, 9A, and 11A), and two β subunit genes (SCN1B and 2B). Our results demonstrate that human PASMC express TTX-sensitive voltage-gated Na+ channels. Their physiological functions remain unresolved, although our data suggest that Na+ channel activity does not directly influence membrane potential, intracellular Ca2+ release, or proliferation in normal human PASMC. Whether their expression and/or activity are heightened in the pathological state is discussed.