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Find video protocols related to scientific articles indexed in Pubmed.
A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 08-29-2014
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Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{?[2-(di-n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs.
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Functional and pharmacological analysis of cardiomyocytes differentiated from human peripheral blood mononuclear-derived pluripotent stem cells.
Stem Cell Reports
PUBLISHED: 07-08-2014
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Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.
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Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.
J. Biol. Chem.
PUBLISHED: 04-01-2014
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Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.
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Malfolded protein structure and proteostasis in lung diseases.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 02-26-2014
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Recent discoveries indicate that disorders of protein folding and degradation play a particularly important role in the development of lung diseases and their associated complications. The overarching purpose of the National Heart, Lung, and Blood Institute workshop on "Malformed Protein Structure and Proteostasis in Lung Diseases" was to identify mechanistic and clinical research opportunities indicated by these recent discoveries in proteostasis science that will advance our molecular understanding of lung pathobiology and facilitate the development of new diagnostic and therapeutic strategies for the prevention and treatment of lung disease. The workshop's discussion focused on identifying gaps in scientific knowledge with respect to proteostasis and lung disease, discussing new research advances and opportunities in protein folding science, and highlighting novel technologies with potential therapeutic applications for diagnosis and treatment.
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Chaperones and cardiac misfolding protein diseases.
Curr. Protein Pept. Sci.
PUBLISHED: 02-13-2014
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Cardiomyocytes are best known for their spontaneous beating activity, large cell size, and low regenerative capacity during adulthood. The mechanical activity of cardiomyocytes depends on a sophisticated contractile apparatus comprised of sarcomeres whose rhythmic contraction relies on Ca(2+) transients with a high level of energy consumption. Hence the proper folding and assembly of the sarcomeric and other accessory proteins involved in those diverse functions (i.e., structural, mechanical, energy exchange and production) is critical for muscle mechanics. Chaperone proteins assist other polypeptides to reach their proper conformation, activity and/or location. Consequently, chaperone-like functions are important for the healthy heart but assume greater relevance during cardiac diseases when such chaperone proteins are recruited: 1) to protect cardiac cells against adverse effects during the pathological transition, and 2) to mitigate certain pathogenic mechanisms per se. Protein misfolding is observed as a consequence of inappropriate intracellular environment with acquired conditions (e.g., ischemia/reperfusion and redox imbalance) or because of mutations, which can modify primary to quaternary protein structures. In this review, we discuss the importance of cardiac chaperones while emphasizing the genetic origin (modification of gene/protein sequence) of cardiac protein misfolding and their consequences on the cardiomyocytes leading to organ dysfunction and failure.
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The NADPH metabolic network regulates human ?B-crystallin cardiomyopathy and reductive stress in Drosophila melanogaster.
PLoS Genet.
PUBLISHED: 06-01-2013
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Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryAB(R120G) missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cells primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryAB(R120G) pathology, confirming the link between NADP/H metabolism and CryAB.
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Modeling human protein aggregation cardiomyopathy using murine induced pluripotent stem cells.
Stem Cells Transl Med
PUBLISHED: 02-19-2013
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Several mutations in ?B-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and ?-skeletal ?-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations.
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ROS Stress Resets Circadian Clocks to Coordinate Pro-Survival Signals.
PLoS ONE
PUBLISHED: 01-01-2013
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Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H2O2 -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) -mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.
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Effects of redox state on the efficient uptake of cell permeable Peptide in Mammalian cells.
Open Biochem J
PUBLISHED: 01-01-2013
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We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC). The FRET peptide construct was easily detected in cells cultured in 96-well plates using a plate-reader. Treatment of cells with various classes of reducing or oxidizing agents resulted in an increase or decrease in FAM fluorescence, respectively. Changes in FAM fluorescence correlated significantly with redox-sensitive green fluorescent protein ratios in cells treated with hydrogen peroxide but not NAC. Detection of relative changes in cellular redox state was enhanced by the fact that uptake of the cell-penetrating peptide occurred more quickly in relatively reduced compared with oxidized cells. We conclude that cell-penetrating peptides coupled via disulfide bonds to detectable cargo is a novel and specific approach for assessment of relative changes in cellular thiol redox state.
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Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.
FASEB J.
PUBLISHED: 12-27-2011
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To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss- and gain-of-function GSH content in vitro. We monitored the redox events in the cytosol and mitochondria using reduction-oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H(2)O(2) or the Trx reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, E(hc) (GSSG/2GSH), respectively. In contrast, N-acetyl-L-cysteine (NAC) treatment in H9c2 cells, or overexpression of either the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) or GCL modifier subunit (GCLM) in human embryonic kidney 293 T (HEK293T) cells, led to 3- to 4-fold increase of GSH and caused 7 or 12 mV more reducing E(hc), respectively. This condition paradoxically increased the level of mitochondrial oxidation, as demonstrated by redox shifts in mitochondrial roGFP and Trx2. Lastly, either NAC treatment (EC(50) 4 mM) or either GCLC or GCLM overexpression exhibited increased cytotoxicity and the susceptibility to the more reducing milieu was achieved at decreased levels of ROS. Taken together, our findings reveal a novel mechanism by which GSH-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.
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Proteostasis and REDOX state in the heart.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 10-14-2011
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Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed "proteostasis." Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans.
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An economic analysis of dose dense weekly paclitaxel plus carboplatin versus every-3-week paclitaxel plus carboplatin in the treatment of advanced ovarian cancer.
Gynecol. Oncol.
PUBLISHED: 07-15-2011
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Compared with every-3-week paclitaxel (q3T) plus carboplatin, dose-dense weekly paclitaxel (ddT) plus carboplatin improved the survival of ovarian cancer patients. We performed a cost analysis comparing these two regimens.
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Synchronization of circadian Per2 rhythms and HSF1-BMAL1:CLOCK interaction in mouse fibroblasts after short-term heat shock pulse.
PLoS ONE
PUBLISHED: 04-08-2011
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Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems.
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HSF1-dependent upregulation of Hsp70 by sulfhydryl-reactive inducers of the KEAP1/NRF2/ARE pathway.
Chem. Biol.
PUBLISHED: 03-28-2011
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The KEAP1/NRF2/ARE pathway and the heat shock response are inducible cytoprotective systems regulated by transcription factors NRF2 and HSF1, respectively. We report that structurally distinct small molecule NRF2 activators, all of which react with sulfhydryl groups but differ in potency by 15,000-fold, upregulate Hsp70, a prototypic HSF1-dependent gene. Hsp70 upregulation requires HSF1 but is NRF2 independent. We further demonstrate that a sulfoxythiocarbamate inducer conjugates to the negative regulator of HSF1, Hsp90. The differential concentration dependence of the two responses suggests that activation of NRF2 precedes that of HSF1: the KEAP1/NRF2/ARE pathway is at the forefront of cellular defense, protecting against instant danger; the heat shock response closely follows to resolve subsequent potentially devastating damage, saving the proteome. This uncovered duality undoubtedly contributes to the cytoprotective effects of such molecules in models of carcinogenesis, cardiovascular disease, and neurodegeneration.
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Sustained activation of nuclear erythroid 2-related factor 2/antioxidant response element signaling promotes reductive stress in the human mutant protein aggregation cardiomyopathy in mice.
Antioxid. Redox Signal.
PUBLISHED: 02-02-2011
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Inheritable missense mutations in small molecular weight heat-shock proteins (HSP) with chaperone-like properties promote self-oligomerization, protein aggregation, and pathologic states such as hypertrophic cardiomyopathy in humans. We recently described that human mutant ?B-crystallin (hR120GCryAB) overexpression that caused protein aggregation cardiomyopathy (PAC) was genetically linked to dysregulation of the antioxidant system and reductive stress (RS) in mice. However, the molecular mechanism that induces RS remains only partially understood. Here we define a critical role for the regulatory nuclear erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein (Keap1) pathway--the master transcriptional controller of antioxidants, in the pathogenesis of PAC and RS. In myopathic mice, increased reactive oxygen species signaling during compensatory hypertrophy (i.e., 3 months) was associated with upregulation of key antioxidants in a manner consistent with Nrf2/antioxidant response element (ARE)-dependent transactivation. In transcription factor assays, we further demonstrate increased binding of Nrf2 to ARE during the development of cardiomyopathy. Of interest, we show that the negative regulator Keap1 was predominantly sequestrated in protein aggregates (at 6 months), suggesting that sustained nuclear translocation of activated Nrf2 may be a contributing mechanism for RS. Our findings implicate a novel pathway for therapeutic targeting and abrogating RS linked to experimental cardiomyopathy in humans. Antioxid.
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Endometrial cancer surgery in Arizona: a statewide analysis of access to care.
Gynecol. Oncol.
PUBLISHED: 01-05-2011
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To investigate access to surgical care for endometrial cancer in Arizona.
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Selective degradation of aggregate-prone CryAB mutants by HSPB1 is mediated by ubiquitin-proteasome pathways.
J. Mol. Cell. Cardiol.
PUBLISHED: 06-06-2010
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Disease-causing mutations of genes encoding small MW heat shock proteins (sHSPs) constitute a growing family of inherited myofibrillar disorders. In the present work, we found that three structurally-distinct CryAB mutants R120G, 450delA and 464delCT are mostly present in the detergent-insoluble fractions when overexpressed in H9c2 rat heart cells. We found that either over-expression or knockdown of HSPB1, a related sHSP, affects the solubility, stability, and degradation of aggregation-prone CryAB mutants. HSPB1 overexpression has negligible effects on the solubility and protein aggregates of either R120G and/or 450delA but increased the solubility and prevented formation of 464delCT aggregates. HSPB1 knockdown decreased solubility and increased protein aggregates of all CryAB mutants, indicating a key role for HSPB1 in clearance of CryAB mutants under basal conditions. We provide four lines of evidence that such selective clearance of R120G, 450delA and 464delCT mutants by HSPB1 is mediated by the ubiquitin-proteasome system (UPS). First, we found that treatment with the proteasome inhibitors increased the levels of all CryAB mutants. Second, R120G and 450delA overexpression corresponded to the accumulation of their specific ubiquitin conjugates in H9c2 cells. Third, HSPB1 knockdown directly increased the levels of all polyubiquitin conjugates. And fourth, the selective attenuation of 464delCT expression by HSPB1 over-expression was abrogated by the proteasome inhibition. We conclude that such selective interactions between CryAB mutants and HSPB1 overexpression might have important implications for the clinical manifestations and potential treatment.
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Involvement of reductive stress in the cardiomyopathy in transgenic mice with cardiac-specific overexpression of heat shock protein 27.
Hypertension
PUBLISHED: 05-03-2010
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Oxidative stress plays an important role in cardiac diseases, which has been well demonstrated, whereas the role of reductive stress has been poorly investigated. We and others have shown previously that heat shock protein 27 (Hsp27) plays a role as an antioxidant. To investigate whether overexpression of Hsp27 could lead to reductive stress and result in cardiomyopathy, we generated transgenic mice with different expression levels of Hsp27. We observed that transgenic mice with high levels of Hsp27 developed cardiomyopathy. The myopathic hearts were under reductive stress, which was evidenced by an increased ratio of reduced glutathione/oxidized glutathione and a decreased level of reactive oxygen species. In addition, upregulated glutathione peroxidase 1 and decreased iron content were revealed in the myopathic hearts. More importantly, inhibition of glutathione peroxidase 1 significantly attenuated the development of cardiomyopathy. The data indicate that the Hsp27-induced cardiomyopathy could be attributed to, at least in part, upregulation of glutathione peroxidase 1. Our findings suggest that reductive stress plays an important role in the development of cardiomyopathy and that Hsp27 may serve as a potential target for the treatment of patients with cardiomyopathy.
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Febrile-range temperature modifies cytokine gene expression in LPS-stimulated macrophages by differentially modifying NF-{kappa}B recruitment to cytokine gene promoters.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 10-21-2009
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We previously showed that exposure to febrile-range temperatures (FRT, 39.5-40 degrees C) reduces LPS-induced TNF-alpha expression, in part through the direct interaction of heat shock factor-1 (HSF1) with the TNF-alpha gene promoter. However, it is not known whether exposure to FRT also modifies more proximal LPS-induced signaling events. Using HSF1-null mice, we confirmed that HSF1 is required for FRT-induced repression of TNF-alpha in vitro by LPS-stimulated bone marrow-derived macrophages and in vivo in mice challenged intratracheally with LPS. Exposing LPS-stimulated RAW 264.7 mouse macrophages to FRT reduced TNF-alpha expression while increasing IL-1beta expression despite the two genes sharing a common myeloid differentiation protein-88 (MyD88)-dependent pathway. Global activation of the three LPS-induced signaling intermediates that lead to cytokine gene expression, ERK and p38 MAPKs and NF-kappaB, was not affected by exposing RAW 264.7 cells to FRT as assessed by ERK and p38 phosphorylation and NF-kappaB in vitro DNA-binding activity and activation of a NF-kappaB-dependent synthetic promoter. However, chromatin immunoprecipitation (ChIP) analysis demonstrated that exposure to FRT reduced LPS-induced recruitment of NF-kappaB p65 to the TNF-alpha promoter while simultaneously increasing its recruitment to the IL-1beta promoter. These data suggest that FRT exerts its effects on cytokine gene expression in a gene-specific manner through distal effects on promoter activation rather than proximal receptor activation and signal transduction.
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Antiangiogenic agents as a maintenance strategy for advanced epithelial ovarian cancer.
Crit. Rev. Oncol. Hematol.
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Bevacizumab is the first antiangiogenic agent to have demonstrated benefit as first-line and maintenance therapy in epithelial ovarian cancer (EOC), with the Gynecologic Oncology Group 218 and ICON 7 phase III trials revealing significantly prolonged progression-free survival (PFS) for carboplatin/paclitaxel plus bevacizumab followed by bevacizumab maintenance versus carboplatin/paclitaxel alone. Results are forthcoming from several phase III maintenance trials of investigational antiangiogenic agents, each evaluating PFS as the primary endpoint: AGO-OVAR12/LUME-Ovar1 (nintedanib [BIBF 1120]), AGO-OVAR16 (pazopanib), and TRINOVA-1, -2, and -3 (AMG 386). Here we review available data and ongoing clinical trials of investigational antiangiogenic agents as maintenance therapy for EOC. Current controversies, including uncertainties regarding the (1) most appropriate clinical trial endpoints, (2) optimal dosing, duration, and timing of therapy (e.g., with first-line chemotherapy and/or as maintenance monotherapy), and (3) feasibility, tolerability, and cost of adding these agents to platinum/taxane regimens are also highlighted.
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Reductive stress linked to small HSPs, G6PD, and Nrf2 pathways in heart disease.
Antioxid. Redox Signal.
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Aerobic organisms must exist between the dueling biological metabolic processes for energy and respiration and the obligatory generation of reactive oxygen species (ROS) whose deleterious consequences can reduce survival. Wide fluctuations in harmful ROS generation are circumvented by endogenous countermeasures (i.e., enzymatic and nonenzymatic antioxidants systems) whose capacity decline with aging and are enhanced by disease states.
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HSPB2 is dispensable for the cardiac hypertrophic response but reduces mitochondrial energetics following pressure overload in mice.
PLoS ONE
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CryAB (HspB5) and HspB2, two small heat shock genes located adjacently in the vertebrate genome, are hypothesized to play distinct roles. Mice lacking both cryab and hspb2 (DKO) are viable and exhibit adult-onset degeneration of skeletal muscle but confounding results from independent groups were reported for cardiac responses to different stressful conditions (i.e., ischemia/reperfusion or pressure overload). To determine the specific requirements of HSPB2 in heart, we generated cardiac-specific HSPB2 deficient (HSPB2cKO) mice and examined their cardiac function under basal conditions and following cardiac pressure overload.
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Small heat shock proteins in redox metabolism: implications for cardiovascular diseases.
Int. J. Biochem. Cell Biol.
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A timely review series on small heat shock proteins has to appropriately examine their fundamental properties and implications in the cardiovascular system since several members of this chaperone family exhibit robust expression in the myocardium and blood vessels. Due to energetic and metabolic demands, the cardiovascular system maintains a high mitochondrial activity but irreversible oxidative damage might ensue from increased production of reactive oxygen species. How equilibrium between their production and scavenging is achieved becomes paramount for physiological maintenance. For example, heat shock protein B1 (HSPB1) is implicated in maintaining this equilibrium or redox homeostasis by upholding the level of glutathione, a major redox mediator. Studies of gain or loss of function achieved by genetic manipulations have been highly informative for understanding the roles of those proteins. For example, genetic deficiency of several small heat shock proteins such as HSPB5 and HSPB2 is well-tolerated in heart cells whereas a single missense mutation causes human pathology. Such evidence highlights both the profound genetic redundancy observed among the multigene family of small heat shock proteins while underscoring the role proteotoxicity plays in driving disease pathogenesis. We will discuss the available data on small heat shock proteins in the cardiovascular system, redox metabolism and human diseases. From the medical perspective, we envision that such emerging knowledge of the multiple roles small heat shock proteins exert in the cardiovascular system will undoubtedly open new avenues for their identification and possible therapeutic targeting in humans. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.
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Trabectedin as a new chemotherapy option in the treatment of relapsed platinum sensitive ovarian cancer.
Curr. Pharm. Des.
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Trabectedin (ET-743, Yondelis®) is a novel marine antineoplastic alkaloid with a unique mechanism of action. The active substance trabectedin, a tetrahydroisoquinoline alkaloid, is a natural product originally isolated from the Caribbean sea squirt, Ecteinascidia turbinata and is currently manufactured by total synthesis. Trabectedin is licensed by the Spanish pharmaceutical drug company, PharmaMar and co-developed by Johnson & Johnson Pharmaceutical Research and Development, L.L.C., pursuant to a licensing agreement with PharmaMar. Trabectedin is the first anticancer marine-derived drug to be approved by the European Union. In 2007, trabectedin obtained marketing authorization from the European Commission and in many other countries worldwide for the treatment of patients with advanced soft tissue sarcoma (STS) after failure of anthracyclines and ifosfamide, or for those patients who are unsuitable to receive these agents. Based on the recently reported results of a large phase III study (OVA-301) comparing pegylated liposomal doxorubicin (PLD) alone with a combination of PLD and trabectedin in patients with recurrent ovarian cancer, in 2009 the European Commission granted marketing authorization for trabectedin combined with PLD for the treatment of patients with relapsed platinum-sensitive ovarian cancer. The results from OVA-301 showed that the combination of trabectedin and PLD improves progression-free survival and overall response rate over PLD alone with acceptable tolerance in the second-line treatment of recurrent ovarian cancer. In addition, an enhanced activity of trabectedin combined with PLD was observed in platinum sensitive patients, especially in those with a platinum-free interval ranging from 6 to 12 months. Overall, trabectedin-induced toxicities are mainly hematological and hepatic, with grade 3/4 neutropenia and thrombocytopenia observed in approximately 50% and 13% of patients, respectively, and grade 3/4 elevation of liver aminotransferases observed in 40-50% of patients treated with trabectedin. Current efforts are focused on the evaluation of the role of trabectedin in prolonging the platinum-free interval and the identification of predictive factors for patients treated with trabectedin as well as in the development of new trabectedin-based combinations.
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The heat shock transcription factor Hsf1 is downregulated in DNA damage-associated senescence, contributing to the maintenance of senescence phenotype.
Aging Cell
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Heat shock response (HSR) that protects cells from proteotoxic stresses is downregulated in aging, as well as upon replicative senescence of cells in culture. Here we demonstrate that HSR is suppressed in fibroblasts from the patients with segmental progerioid Werner Syndrome, which undergo premature senescence. Similar suppression of HSR was seen in normal fibroblasts, which underwent senescence in response to DNA damaging treatments. The major DNA-damage-induced signaling (DDS) pathways p53-p21 and p38-NF-kB-SASP contributed to the HSR suppression. The HSR suppression was associated with inhibition of both activity and transcription of the heat shock transcription factor Hsf1. This inhibition in large part resulted from the downregulation of SIRT1, which in turn was because of decrease in the expression of the translation regulator HuR. Importantly, we uncovered a positive feedback regulation, where suppression of Hsf1 further activates the p38-NF-?B-SASP pathway, which in turn promotes senescence. Overexpression of Hsf1 inhibited the p38-NF?B-SASP pathway and partially relieved senescence. Therefore, downregulation of Hsf1 plays an important role in the development or in the maintenance of DNA damage signaling-induced cell senescence.
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Aggregate-prone R120GCRYAB triggers multifaceted modifications of the Thioredoxin System.
Antioxid. Redox Signal.
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Aims: The human mutation R120G in the ?B-crystallin causes a multisystemic disease characterized by hypertrophic cardiomyopathy and cytoplasmic protein aggregates. In transgenic mice, human R120GCRYAB (hR120GTg) expression in heart sequentially modifies the REDOX status, in part by the activation of the nuclear factor, erythroid derived 2, like 2 (Nrf2). Thioredoxin System (TS) components are NRF2 target genes so it could be hypothesized that TS was affected in hR120GTg mice. Results: Transgenic hearts overexpressed thioredoxin1 (Trx1), which was identified by Isotope Coded Affinity Tag-Mass Spectrometry (ICAT), among hundreds of peptides displaying an increased reduced/oxidized ratio. Coupled to this higher level of reduced cysteines, the activity of thioredoxin reductase 1 (TrxR1) was augmented by 2.5 fold. Combining mutiple experimental approaches, the enzymatic regulation of TrxR1 by a HDAC3-dependent level of acetylation was confirmed. In vitro and in vivo functional tests established that TrxR1 activity is required to mitigate aggregate development and this could be mediated by BAG3 as a potential TS substrate. Innovation and Conclusions: This study uncovers the compartmentalized changes and the involvement of TS in the cardiac stress response elicited by misfolded proteins such as R120GCRYAB. Our work suggests that R120GCRYAB triggers a defensive pathway acting through the newly identified interacting partners HDAC3, TrxR1 and BAG3 to counter aggregate growth. Therefore, those interactors may function as modifier genes contributing to the variable onset and expressivity of such human diseases. Furthermore, our work underscores the potential organismal effects of pharmacological interventions targeting TS and HDAC.
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