Cardiomyocyte-specific Peli1 contributes to the pressure overload-induced cardiac fibrosis through miR-494-3p-dependent exosomal communication.

Cardiac fibrosis is an essential pathological process in pressure overload (PO)-induced heart failure. Recently, myocyte-fibroblast communication is proven to be critical in heart failure, in which, pathological growth of cardiomyocytes (CMs) may promote fibrosis via miRNAs-containing exosomes (Exos). Peli1 regulates the activation of NF-κB and AP-1, which has been demonstrated to engage in miRNA transcription in cardiomyocytes. Therefore, we hypothesized that Peli1 in CMs regulates the activation of cardiac fibroblasts (CFs) through an exosomal miRNA-mediated paracrine mechanism, thereby promoting cardiac fibrosis. We found that CM-conditional deletion of Peli1 improved PO-induced cardiac fibrosis. Moreover, Exos from mechanical stretch (MS)-induced WT CMs (WT MS-Exos) promote activation of CFs, Peli1-/- MS-Exos reversed it. Furthermore, miRNA microarray and qPCR analysis showed that miR-494-3p was increased in WT MS-Exos while being down regulated in Peli1-/- MS-Exos. Mechanistically, Peli1 promoted miR-494-3p expression via NF-κB/AP-1 in CMs, and then miR-494-3p induced CFs activation by inhibiting PTEN and amplifying the phosphorylation of AKT, SMAD2/3, and ERK. Collectively, our study suggests that CMs Peli1 contributes to myocardial fibrosis via CMs-derived miR-494-3p-enriched exosomes under PO, and provides a potential exosomal miRNA-based therapy for cardiac fibrosis.

Large-scale networks changes in Wilson's disease associated with neuropsychiatric impairments: a resting-state functional magnetic resonance imaging study.

BACKGROUND: In Wilson's disease (WD) patients, network connections across the brain are disrupted, affecting multidomain function. However, the details of this neuropathophysiological mechanism remain unclear due to the rarity of WD. In this study, we aimed to investigate alterations in brain network connectivity at the whole-brain level (both intra- and inter-network) in WD patients through independent component analysis (ICA) and the relationship between alterations in these brain network functional connections (FCs) and clinical neuropsychiatric features to understand the underlying pathophysiological and central compensatory mechanisms. METHODS: Eighty-five patients with WD and age- and sex-matched 85 healthy control (HC) were recruited for resting-state functional magnetic resonance imaging (rs-fMRI) scanning. We extracted the resting-state networks (RSNs) using the ICA method, analyzed the changes of FC in these networks and the correlation between alterations in FCs and clinical neuropsychiatric features. RESULTS: Compared with HC, WD showed widespread lower connectivity within RSNs, involving default mode network (DMN), frontoparietal network (FPN), somatomotor network (SMN), dorsal attention network (DAN), especially in patients with abnormal UWDRS scores. Furthermore, the decreased FCs in the left medial prefrontal cortex (L_ MPFC), left anterior cingulate gyrus (L_ACC), precuneus (PCUN)within DMN were negatively correlated with the Unified Wilson's Disease Rating Scale-neurological characteristic examination (UWDRS-N), and the decreased FCs in the L_MPFC, PCUN within DMN were negatively correlated with the Unified Wilson's Disease Rating Scale-psychiatric symptoms examination (UWDRS-P). We additionally discovered that the patients with WD exhibited significantly stronger FC between the FPN and DMN, between the DAN and DMN, and between the FPN and DAN compared to HC. CONCLUSIONS: We have provided evidence that WD is a disease with widespread dysfunctional connectivity in resting networks in brain, leading to neurological features and psychiatric symptoms (e.g. higher-order cognitive control and motor control impairments). The alter intra- and inter-network in the brain may be the neural underpinnings for the neuropathological symptoms and the process of injury compensation in WD patients.

Peli1 contributes to myocardial ischemia/reperfusion injury by impairing autophagy flux via its E3 ligase mediated ubiquitination of P62.

Autophagy flux is impaired during myocardial ischemia/reperfusion (M-I/R) via the accumulation of autophagosome and insufficient clearance, which exacerbates cardiomyocyte death. Peli1 (Pellion1) is a RING finger domain-containing ubiquitin E3 ligase that could catalyze the polyubiquitination of substrate proteins. Peli1 has been demonstrated to play an important role in ischemic cardiac diseases. However, little is known about whether Peli1 is involved in the regulation of autophagy flux during M-I/R. The present study investigated whether M-I/R induced impaired autophagy flux could be mediated through Peli1 dependent mechanisms. We induced M-I/R injury in wild type (WT) and Peli1 knockout mice and observed that M-I/R significantly decreased cardiac function that was associated with increased cardiac Peli1 expression and upregulated autophagy-associated protein LC3II and P62. In contrast, Peli1 knockout mice exhibited significant improvement of M-I/R induced cardiac dysfunction and decreased LC3II and P62 expression. Besides, inhibitors of autophagy also increased the infarct size in Peli1 knockout mice after 24 h of reperfusion. Mechanistic studies demonstrated that in vivo I/R or in vitro hypoxia/reoxygenation (H/R) markedly increased the Peli1 E3 ligase activity which directly promoted the ubiquitination of P62 at lysine(K)7 via K63-linkage to inhibit its dimerization and autophagic degradation. Co-immunoprecipitation and GST-pull down assay indicated that Peli1 interacted with P62 via the Ring domain. In addition, Peli1 deficiency also decreased cardiomyocyte apoptosis. Together, our work demonstrated a critical link between increased expression and activity of Peli1 and autophagy flux blockage in M-I/R injury, providing insight into a promising strategy for treating myocardium M-I/R injury.

Triad3A attenuates pathological cardiac hypertrophy involving the augmentation of ubiquitination-mediated degradation of TLR4 and TLR9.

Activation of TLRs mediated the NF-κB signaling pathway plays an important pathophysiological role in cardiac hypertrophy. Triad3A, a ubiquitin E3 ligase, has been reported to negatively regulate NF-κB activation pathway via promoting ubiquitination and degradation of TLR4 and TLR9 in innate immune cells. The role of Triad3A in cardiac hypertrophic development remains unknown. The present study investigated whether there is a link between Triad3A and TLR4 and TLR9 in pressure overload induced cardiac hypertrophy. We observed that Triad3A levels were markedly reduced following transverse aortic constriction (TAC) induced cardiac hypertrophy. Similarly, stimulation of neonatal rat cardiac myocytes (NRCMs) with angiotensin-II (Ang II) significantly decreased Triad3A expression. To determine the role of Triad3A in TAC-induced cardiac hypertrophy, we transduced the myocardium with adenovirus expressing Triad3A followed by induction of TAC. We observed that increased expression of Triad3A significantly attenuated cardiac hypertrophy and improved cardiac function. To investigate the mechanisms by which Triad3A attenuated cardiac hypertrophy, we examined the Triad3A E3 ubiquitination on TLR4 and TLR9. We found that Triad3A promoted TLR4 and TLR9 degradation through ubiquitination. Triad3A mediated TLR4 and TLR9 degradation resulted in suppression of NF-κB activation. Our data suggest that Triad3A plays a protective role in the development of cardiac hypertrophy, at least through catalyzing ubiquitination-mediated degradation of TLR4 and TLR9, thus negatively regulating NF-κB activation.

Magnetism and hybrid improper ferroelectricity in LaMO3/YMO3 superlattices.

Using first-principles calculations, we investigate the structural, electronic, and magnetic properties of perovskite LaMO3/YMO3 superlattices (M = Cr, Mn, Co and Ni). It is found that ferroelectricity can emerge in LaMO3/YMO3 superlattices (M = Cr, Mn, Co), allowing them to be promising multiferroic candidates, while no ferroelectricity is found in the LaNiO3/YNiO3 superlattice. The electronic structure calculations indicate that the LaCrO3/YCrO3, LaMnO3/YMnO3, and LaCoO3/YCoO3 superlattices are insulators, and their magnetic ground states exhibit G-type antiferromagnetic (AFM), A-type AFM, and G-type AFM order, respectively, while the LaNiO3/YNiO3 superlattice is however a half-metallic ferromagnet. The electronic structure and magnetic ground state are discussed, based on the projected density of states data and Heisenberg model, respectively, and the magnetic phase transition temperature is evaluated based on mean-field theory. In the meantime, the spontaneous ferroelectric polarization of the LaMO3/YMO3 superlattices (M = Cr, Mn, Co) is determined respectively using the Born effective charge model and Berry phase method, and their hybrid improper ferroelectric character is predicted, with the net polarization mainly from the different displacements of the LaO layers and YO layers along the b-axis. It is suggested that alternative multiferroic materials can be obtained by properly designing superlattices that consist of two non-polar magnetic materials but exhibit tunable magnetic ground states and transition temperature and hybrid improper ferroelectricity.

HSPA12B promotes functional recovery after ischaemic stroke through an eNOS-dependent mechanism.

Stroke is the leading cause of disability worldwide. HSPA12B, a heat-shock protein recently identified expression specifically in endothelial cells, is able to promote angiogenesis. Here, we have investigated its effects on functional recovery at chronic phase of ischaemic stroke. Ischaemic stroke was induced by 60 min. of middle cerebral artery occlusion in transgenic mice with overexpression of HSPA12B (HSPA12B Tg) and wild-type littermates (WT). HSPA12B Tg mice demonstrated a significant higher survival rate than WT mice within 28 days post-stroke. Significant improved neurological functions, increased spontaneous locomotor activity and decreased anxiety were detected inHSPA12B Tg mice compared with WT controls within 21 days post-stroke. Stroke-induced hippocampal degeneration was attenuated in HSPA12B Tg mice examined at day 28 post-stroke. Interestingly, HSPA12B Tg mice showed enhanced peri-infarct angiogenesis (examined 28 days post-stroke) and hippocampal neurogenesis (examined 7 days post-stroke), respectively, compared to WT mice. The stroke-induced eNOS phosphorylation and TGF-ß1 expression were augmented in HSPA12B Tg mice. However, administration with eNOS inhibitor L-NAME diminished the HSPA12B-induced protection in neurological functional recovery and mice survival post-stroke. The data suggest that HSPA12B promoted functional recovery and survival after stroke in an eNOS-dependent mechanism. Targeting HSPA12B expression may have a therapeutic potential for the stroke-evoked functional disability and mortality.

Heat shock protein A12A encodes a novel prosurvival pathway during ischaemic stroke.

Heat shock protein A12A (HSPA12A) is a newly discovered member of the Hsp70 family. The biological characteristics and functional roles of HSPA12A are poorly understood. This study investigated the effects of HSPA12A on ischaemic stroke in mice. Ischaemic stroke was induced by left middle cerebral artery occlusion for 1 h followed by blood reperfusion. We observed that HSPA12A was highly expressed in brain neurons, and neuronal HSPA12A expression was downregulated by ischaemic stroke and stroke-associated risk factors (aging, obesity and hyperglycaemia). To investigate the functional requirement of HSPA12A in protecting ischaemic brain injury, HSPA12A knockout mice (Hspa12a-/-) were generated. Hspa12a-/- mice exhibited an enlarged infarct volume and aggravated neurological deficits compared to their wild-type (WT) littermates after stroke. These aggravations in Hspa12a-/- mice were accompanied by more apoptosis and severer hippocampal morphological abnormalities in ischaemic hemispheres. Long-term examination revealed impaired motor function recovery and neurogenesis in stroke-affected Hspa12a-/- mice compared to stroke-affected WT controls. Significant reduced activation of GSK-3ß/mTOR/p70S6K signalling was also observed in ischaemic hemispheres of Hspa12a-/- mice compared to WT controls. Administration with lithium (non-selective GSK-3ß inhibitor) activated GSK-3ß/mTOR/p70S6K signalling in stroke-affected Hspa12a-/- mice. Notably, lithium administration attenuated the HSPA12A deficiency-induced aggravation in infarct size, neurological deficits and neuronal death in Hspa12a-/- mice after stroke. Altogether, the findings suggest that HSPA12A expression encodes a critical novel prosurvival pathway during ischaemic stroke. We identified HSPA12A as a novel neuroprotective target for stroke patients.

Enhanced Glycolytic Metabolism Contributes to Cardiac Dysfunction in Polymicrobial Sepsis.

Background: Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Methods: Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Results: Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1ß as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Conclusions: Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

The TIR/BB-loop mimetic AS-1 attenuates mechanical stress-induced cardiac fibroblast activation and paracrine secretion via modulation of large tumor suppressor kinase 1.

The TIR/BB-loop mimetic AS-1 has been reported to prevent cardiac hypertrophy by inhibiting interleukin-1 receptor (IL-1R)-mediated myeloid differentiation primary response gene 88 (MyD88)-dependent signaling. To date, it remains unknown whether and if so how AS-1 contributes to mechanical stress (MS)-induced cardiac fibroblast activation, a key process in pressure overload-induced cardiac remodeling and heart failure. Here, we show that phosphorylation and expression of large tumor suppressor kinase 1 (LATS1), a key molecule in the Hippo-Yes associated protein (YAP) signaling pathway, were down-regulated in primary neonatal rat cardiac fibroblasts (NRCFs) in response to MS and in the hearts of mice subjected to transverse aortic constriction (TAC) procedure; AS-1 treatment was able to restore LATS1 phosphorylation and expression both in vitro and in vivo. AS-1 treatment suppressed the induction of proliferation, differentiation and collagen synthesis in response to MS in NRCFs. AS-1 also ameliorated cardiomyocyte hypertrophy and apoptosis through dampening paracrine secretion of stretched cardiac fibroblasts. In mice, AS-1 treatment could protect against TAC-induced cardiac hypertrophy, myocardial fibrosis and heart failure. Of note, LATS1 depletion using siRNA completely abrogated the inhibitory effects of AS-1 on NRCFs under MS including accelerated proliferation, differentiation, enhanced ability to produce collagen and augmented paracrine secretion of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) to induce cardiomyocyte hypertrophy. Therefore, our results delineate a previously unrecognized role for LATS1 in cardiac fibroblast to mediate the beneficial effects of AS-1 in preventing pressure overload-induced cardiac remodeling and heart failure.

Attenuation of Cardiac Dysfunction in Polymicrobial Sepsis by MicroRNA-146a Is Mediated via Targeting of IRAK1 and TRAF6 Expression.

Cardiac dysfunction is a major consequence of sepsis/septic shock and contributes to the high mortality of sepsis. Innate and inflammatory responses mediated by TLRs play a critical role in sepsis-induced cardiac dysfunction. MicroRNA-146 (miR-146) was first identified as a negative regulator in innate immune and inflammatory responses induced by LPS. This study examined whether miR-146a will have a protective effect on sepsis-induced cardiac dysfunction. Lentivirus-expressing miR-146a (LmiR-146a) or lentivirus-expressing scrambled miR (LmiR-control) was delivered into the myocardium via the right carotid artery. Seven days after transfection, mice were subjected to cecal ligation and puncture (CLP). Untransfected mice were also subjected to CLP-induced sepsis. Cardiac function was examined by echocardiography before and 6 h after CLP. In vitro studies showed that increased miR-146a levels suppress LPS-induced IκBα phosphorylation and inflammatory cytokine production in both H9C2 cardiomyocytes and J774 macrophages. In vivo transfection of LmiR-146a attenuated sepsis-induced cardiac dysfunction. The values for percent ejection fraction and percent fractional shortening in LmiR-146a-transfected CLP mice were significantly greater than in untransfected CLP control. LmiR-146a transfection prevented sepsis-induced NF-κB activity, suppressed IRAK and TRAF6 expression in the myocardium, and attenuated sepsis-induced inflammatory cytokine production in both plasma and peritoneal fluid. In addition, LmiR-146a transfection decreased sepsis-induced infiltration of neutrophils and macrophages into the myocardium. LmiR-146a can also transfect macrophages in the periphery. We conclude that miR-146a attenuates sepsis-induced cardiac dysfunction by preventing NF-κB activation, inflammatory cell infiltration, and inflammatory cytokine production via targeting of IRAK and TRAF6 in both cardiomyocytes and inflammatory monocytic cells.

Study on Lesion Assessment of Cerebello-Thalamo-Cortical Network in Wilson's Disease with Diffusion Tensor Imaging.

Wilson's disease (WD) is a genetic disorder of copper metabolism with pathological copper accumulation in the brain and any other tissues. This article aimed to assess lesions in cerebello-thalamo-cortical network with an advanced technique of diffusion tensor imaging (DTI) in WD. 35 WD patients and 30 age- and sex-matched healthy volunteers were recruited to accept diffusion-weighted images with 15 gradient vectors and conventional magnetic resonance imaging (MRI). The DTI parameters, including fractional anisotropy (FA) and mean diffusion (MD), were calculated by diffusion kurtosis estimator software. After registration, patient groups with FA mappings and MD mappings and normal groups were compared with 3dttest and receiver-operating characteristic (ROC) curve analysis, corrected with FDR simulations (p = 0.001, α = 0.05, cluster size = 326). We found that the degree of FA increased in the bilateral head of the caudate nucleus (HCN), lenticular nucleus (LN), ventral thalamus, substantia nigra (SN), red nucleus (RN), right dentate nucleus (DN), and decreased in the mediodorsal thalamus and extensive white matter. The value of MD increased in HCN, LN, SN, RN, and extensive white matter. The technique of DTI provides higher sensitivity and specificity than conventional MRI to detect Wilson's disease. Besides, lesions in the basal ganglia, thalamus, and cerebellum might disconnect the basal ganglia-thalamo-cortical circuits or dentato-rubro-thalamic (DRT) track and disrupt cerebello-thalamo-cortical network finally, which may cause clinical extrapyramidal symptoms.

MicroRNA-125b Prevents Cardiac Dysfunction in Polymicrobial Sepsis by Targeting TRAF6-Mediated Nuclear Factor κB Activation and p53-Mediated Apoptotic Signaling.

BACKGROUND: This study examined the effect of microRNA-125b (miR-125b) on sepsis-induced cardiac dysfunction. METHODS: Mouse hearts were transfected with lentivirus expressing miR-125b (LmiR-125b) 7 days before cecal ligation and puncture (CLP)-induced sepsis. Cardiac function was examined by echocardiography before and 6 hours after CLP (n = 6/group). Survival was monitored following CLP-induced sepsis (n = 12/group). RESULTS: LmiR-125b transfection significantly attenuated cardiac dysfunction due to CLP-induced sepsis. Fractional shortening and ejection fraction values were significantly (P < .05) higher in the LmiR-125b-treated CLP group than in the untreated CLP group. Survival outcome in LmiR-125b-transfected septic mice was markedly improved, compared with mice with CLP-induced sepsis. Transfection of LmiR-125b into the heart significantly suppressed the expression of ICAM-1 and VCAM-1, decreased the accumulation of macrophages and neutrophils in the myocardium, and decreased serum levels of tumor necrosis factor α and interleukin 1ß by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated nuclear factor κB (NF-κB) activation. In addition, sepsis-induced myocardial apoptosis was markedly attenuated by LmiR-125b transfection through suppression of p53, Bax, and Bak1 expression. In vitro transfection of endothelial cells with miR-125b mimics attenuate LPS-induced ICAM-1 and VCAM-1 expression by suppressing TRAF6 and NF-κB activation. CONCLUSIONS: Increased myocardial miR-125b expression attenuates sepsis-induced cardiac dysfunction and improves survival. miR-125b may be a target for septic cardiomyopathy.

Cortical Reorganization in Patients Recovered from Bell's Palsy: An Orofacial and Finger Movements Task-State fMRI Study.

Objective. To explore cortical reorganization of patients recovered from Bell's palsy (BP) by task-state functional magnetic resonance imaging (fMRI) during finger and orofacial movements and provide more evidence for acupuncture clinical treatment of BP. Methods. We collected 17 BP patients with complete clinical recovery (BP group) and 20 healthy volunteers (control group) accepted the task-state fMRI scans with lip pursing movements and finger movements, respectively. Results. It was found that there were significant differences of brain functional status between the two groups. Conclusions. The results showed that there was cortical reorganization in the brain of patients recovered from BP after acupuncture treatment, which also suggested the relationship between the hand motor areas and facial motor areas of BP patients.

Attenuation of cardiac dysfunction and remodeling of myocardial infarction by microRNA-130a are mediated by suppression of PTEN and activation of PI3K dependent signaling.

OBJECTIVE: Activation of PI3K/Akt signaling protects the myocardium from ischemia/reperfusion injury. MicroRNAs have been demonstrated to play an important role in the regulation of gene expression at the post-transcriptional level. In this study, we examined whether miR-130a will attenuate cardiac dysfunction and remodeling after myocardial infarction (MI) via PI3K/Akt dependent mechanism. APPROACHES AND RESULTS: To determine the role of miR-130a in the proliferation and migration of endothelial cells, HUVECs were transfected with miR-130a mimics before the cells were subjected to scratch-induced wound injury. Transfection of miR-130a mimics stimulated the migration of endothelial cells into the wound area and increased phospho-Akt levels. To examine the effect of miR-130a on cardiac dysfunction and remodeling after MI, Lentivirus expressing miR-130a (LmiR-130a) was delivered into mouse hearts seven days before the mice were subjected to MI. Cardiac function was assessed by echocardiography before and for up to 21 days after MI. Ejection fraction (EF%) and fractional shortening (FS%) in the LmiR-130a transfected MI hearts were significantly greater than in LmiR-control and untransfected control MI groups. LmiR-130a transfection increased capillary number and VEGF expression, and decreased collagen deposition in the infarcted myocardium. Importantly, LmiR-130a transfection significantly suppressed PTEN expression and increased the levels of phosphorylated Akt in the myocardium. However, treatment of LmiR-130a-transfected mice with LY294002, a PI3K inhibitor, completely abolished miR-130a-induced attenuation of cardiac dysfunction after MI. CONCLUSIONS: miR-130a plays a critical role in attenuation of cardiac dysfunction and remodeling after MI. The mechanisms involve activation of PI3K/Akt signaling via suppression of PTEN expression.

Pellino1-mediated TGF-ß1 synthesis contributes to mechanical stress induced cardiac fibroblast activation.

Activation of cardiac fibroblasts is a key event in the progression of cardiac fibrosis that leads to heart failure. However, the molecular mechanisms underlying mechanical stress-induced cardiac fibroblast activation are complex and poorly understood. This study demonstrates that Pellino1, an E3 ubiquitin ligase, was activated in vivo in pressure overloaded rat hearts and in cultured neonatal rat cardiac fibroblasts (NRCFs) exposed to mechanical stretch in vitro. Suppression of the expression and activity of Pellino1 by adenovirus-mediated delivery of shPellino1 (adv-shpeli1) attenuated pressure overload-induced cardiac dysfunction and cardiac hypertrophy and decreased cardiac fibrosis in rat hearts. Transfection of adv-shpeli1 also significantly attenuated mechanical stress-induced proliferation, differentiation and collagen synthesis in NRCFs. Pellino1 silencing also abrogated mechanical stretch-induced polyubiquitination of tumor necrosis factor-alpha receptor association factor-6 (TRAF6) and receptor-interacting protein 1 (RIP1) and consequently decreased the DNA binding activity of nuclear factor-kappa B (NF-κB) in NRCFs. In addition, Pellino1 silencing prevented stretch-induced activation of p38 and activator protein 1 (AP-1) binding activity in NRCFs. Chromatin Immunoprecipitation (ChIP) and luciferase reporter assays showed that Pellino1 silencing prevented the binding of NF-κB and AP-1 to the promoter region of transforming growth factor-ß1 (TGF-ß1) thus dampening TGF-ß1 transactivation. Our data reveal a previously unrecognized role of Pellino1 in extracellular matrix deposition and cardiac fibroblast activation in response to mechanical stress and provides a novel target for treatment of cardiac fibrosis and heart failure.

Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury via TLR3-mediated prevention of Fas/FADD interaction.

Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.

HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells.

Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 µg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor-α was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.

Class III PI3K-mediated prolonged activation of autophagy plays a critical role in the transition of cardiac hypertrophy to heart failure.

Pathological cardiac hypertrophy often leads to heart failure. Activation of autophagy has been shown in pathological hypertrophic hearts. Autophagy is regulated positively by Class III phosphoinositide 3-kinase (PI3K). However, it is unknown whether Class III PI3K plays a role in the transition of cardiac hypertrophy to heart failure. To address this question, we employed a previously established cardiac hypertrophy model in heat shock protein 27 transgenic mice which shares common features with several types of human cardiomyopathy. Age-matched wild-type mice served as control. Firstly, a prolonged activation of autophagy, as reflected by autophagosome accumulation, increased LC3 conversion and decreased p62 protein levels, was detected in hypertrophic hearts from adaptive stage to maladaptive stage. Moreover, morphological abnormalities in myofilaments and mitochondria were presented in the areas accumulated with autophagosomes. Secondly, activation of Class III PI3K Vacuolar protein sorting 34 (Vps34), as demonstrated by upregulation of Vps34 expression, increased interaction of Vps34 with Beclin-1, and deceased Bcl-2 expression, was demonstrated in hypertrophic hearts from adaptive stage to maladaptive stage. Finally, administration with Wortmaninn, a widely used autophagy inhibitor by suppressing Class III PI3K activity, significantly decreased autophagy activity, improved morphologies of intracellular apartments, and most importantly, prevented progressive cardiac dysfunction in hypertrophic hearts. Collectively, we demonstrated that Class III PI3K plays a central role in the transition of cardiac hypertrophy to heart failure via a prolonged activation of autophagy in current study. Class III PI3K may serve as a potential target for the treatment and management of maladaptive cardiac hypertrophy.

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury.

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3(-/-)) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3(-/-) and WT mice were subjected to myocardial ischemia (45min) followed by reperfusion for up to 3days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3(-/-) mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3(-/-) mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1ß) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.

Impaired autophagosome clearance contributes to local anesthetic bupivacaine-induced myotoxicity in mouse myoblasts.

BACKGROUND: The current study examined the role(s) of autophagy in myotoxicity induced by bupivacaine in mouse myoblast C2c12 cells. METHODS: C2c12 cells were treated with bupivacaine. Myotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (n = 3 to 30), live/dead assay (n = 3 to 4), and morphological alterations (n = 3). Autophagosome formation was reflected by microtubule-associated protein light chain 3 conversion (n = 4 to 12) and light chain 3 punctation (n = 4 to 5). Autophagosome clearance was evaluated by p62 protein level (n = 4) and autolysosomes generation (n = 3). RESULTS: Bupivacaine induced significant cell damage. Notably, there was a significant increase in autophagosome generation as evidenced by light chain 3 puncta formation (72.7 ± 6.9 vs. 2.1 ± 1.2) and light chain 3 conversion (2.16 ± 0.15 vs. 0.33 ± 0.04) in bupivacaine-treated cells. Bupivacaine inactivated the protein kinase B/mammalian target of rapamycin/p70 ribosomal protein S6 kinase signaling. However, cellular levels of p62 protein were significantly increased upon bupivacaine treatment (1.29 ± 0.15 vs. 1.00 ± 0.15), suggesting that the drug impaired autophagosome clearance. Further examination revealed that bupivacaine interrupted autophagosome-lysosome fusion (10.87% ± 1.48% vs. 32.94% ± 4.22%). Administration of rapamycin increased autophagosome clearance and, most importantly, improved the survival in bupivacaine-treated cells. However, knockdown of autophagy-related protein 5 (atg5) exacerbated bupivacaine-induced impairment of autophagosome clearance and myotoxicity. CONCLUSIONS: The data suggest that autophagosome formation was induced as a stress response mechanism after bupivacaine challenge; however, autophagosome clearance was impaired due to inadequate autophagosome-lysosome fusion. Therefore, impairment of autophagosome clearance appears to be a novel mechanism underlying bupivacaine-induced myotoxicity.