The therapeutic effect of rosuvastatin on cardiac remodelling from hypertrophy to fibrosis during the end-stage hypertension in rats

End-stage hypertensive heart disease is an increasing cause of cardiac mortality. Therefore, the current study focused on the cardiac remodelling from hypertrophy to fibrosis in old-aged spontaneously hypertensive rats (SHRs), and explored the therapeutic effects of Rosuvastatin and its possible mechanism(s) of action. Spontaneously hypertensive rats at age 52 weeks were randomly divided into three groups, the first two to receive Rosuvastatin at a dose of 20 mg/kg/day and 40 mg/kg/day, respectively, and the third to receive placebo, which was to be compared with Wistar-Kyoto as controls. After 2-month treatment, SBP, heart to body weight ratio (HW/BW%) and echocardiographic features were evaluated, followed by haematoxylin and eosin and Masson trichrome staining in conjunction with qPCR of foetal gene expressions. Transferase-mediated dUTP nick-end labelling assay and immunofluorescent labelling for active caspase-3 were used to detect the apoptotic cardiomyocytes. Signaling pathways involved were examined by using western blot. Old-aged SHR developed end-stage hypertensive heart disease characterized by significant enhancement of HW/BW%, LVAWd and LVPWd, and decreased LVEF and LVFS, accompanied by cardiomyocytes enlargement and fibrosis along with activation of foetal gene programme. Cardiac apoptosis increased significantly during the transition process. Rosuvastatin reduced hypertrophy significantly via AT(1) Receptor-PKCβ2/α-ERK-c-fos pathway; protected myocardium against apoptosis via Akt-FOXO1, Bcl-2 family and survivin pathways and consequently suppressed the caspase-3 activity. The present study revealed that old-aged SHRs developed cardiac remodelling from hypertrophy to fibrosis via cardiac apoptosis during the end stage of hypertensive heart disease. These pathological changes might be the consequence of activation of AT(1) Receptor-PKCβ2/α-ERK-c-fos and AKT-FOXO1/Bcl-2/survivin/Caspase3 signaling. Rosuvastatin effectively attenuated the structural changes by reversing the signaling transductions involved.     

Gallic acid attenuates calcium calmodulin‐dependent kinase II‐induced apoptosis in spontaneously hypertensive rats

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition‐induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti‐cancer, anti‐calcification and anti‐oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase‐3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ‐induced apoptosis.     

A novel TXNIP‐based mechanism for Cx43‐mediated regulation of oxidative drug injury

Gap junctions (GJs) play an important role in the regulation of cell response to many drugs. However, little is known about their mechanisms. Using an in vitro model of cytotoxicity induced by geneticin (G418), we explored the potential signalling mechanisms involved. Incubation of cells with G418 resulted in cell death, as indicated by the change in cell morphology, loss of cell viability and activation of caspase‐3. Before the onset of cell injury, G418 induced reactive oxygen species (ROS) generation, activated oxidative sensitive kinase P38 and caused a shift of connexin 43 (Cx43) from non‐phosphorylated form to hyperphosphorylated form. These changes were largely prevented by antioxidants, suggesting an implication of oxidative stress. Downregulation of Cx43 with inhibitors or siRNA suppressed the expression of thioredoxin‐interacting protein (TXNIP), activated Akt and protected cells against the toxicity of G418. Further analysis revealed that inhibition of TXNIP with siRNA activated Akt and reproduced the protective effect of Cx43‐inhibiting agents, whereas suppression of Akt sensitized cells to the toxicity of G418. Furthermore, interference of TXNIP/Akt also affected puromycin‐ and adriamycin‐induced cell injury. Our study thus characterized TXNIP as a presently unrecognized molecule implicated in the regulatory actions of Cx43 on oxidative drug injury. Targeting Cx43/TXNIP/Akt signalling cascade might be a promising approach to modulate cell response to drugs.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Cardiac β‐adrenergic receptor activation mediates distinct and cell type‐dependent changes in the expression and distribution of connexin 43

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β₂‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β₂‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β₂‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.     

Inhibition of autophagy enhances the effects of the AKT inhibitor MK‐2206 when combined with paclitaxel and carboplatin in BRAF wild‐type melanoma

This study investigates the mechanism of action behind the long‐term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK‐2206 in combination with paclitaxel and carboplatin. Although single agent MK‐2206 inhibited phospho‐AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK‐2206 with paclitaxel and carboplatin was cytotoxic in long‐term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase‐dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti‐oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.     

Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43‐silenced MLO‐Y4 osteocytic (Cx43^def) cells undergo spontaneous cell death in culture through caspase‐3 activation and exhibit increased levels of apoptosis‐related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43^def cell death. Cx43^def cells and bones from old mice exhibit reduced levels of the pro‐survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43^def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43‐expressing cells and miR21 deletion in miR21^fl/fl bones increases apoptosis‐related gene expression, whereas a miR21 mimic prevents Cx43^def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43^def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high‐mobility group box‐1 (HMGB1)], and caspase‐3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43^def cells, which is blocked by antagonizing HMGB1‐RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.     

Cx31.1 expression is modulated in HaCaT cells exposed to UV‐induced damage and scrape‐wounding

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape‐wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape‐wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with ‘diabetic’ conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn‐over around diabetic wounds.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

Follicle‐stimulating hormone promotes age‐related endometrial atrophy through cross‐talk with transforming growth factor beta signal transduction pathway

It is widely believed that endometrial atrophy in postmenopausal women is due to an age‐related reduction in estrogen level. But the role of high circulating follicle‐stimulating hormone (FSH) in postmenopausal syndrome is not clear. Here, we explored the role of high circulating FSH in physiological endometrial atrophy. We found that FSH exacerbated post‐OVX endometrial atrophy in mice, and this effect was ameliorated by lowering FSH with Gonadotrophin‐releasing hormone agonist (GnRHa). In vitro, FSH inhibited endometrial proliferation and promoted the apoptosis of primary cultured endometrial cells in a dose‐dependent manner. In addition, upregulation of caspase3, caspase8, caspase9, autophagy‐related proteins (ATG3, ATG5, ATG7, ATG12 and LC3) and downregulation of c‐Jun were also observed in endometrial adenocytes. Furthermore, smad2 and smad3 showed a time‐dependent activation in endometrial cells which can be partly inhibited by blocking the transforming growth factor beta receptor II (TβRII). In conclusion, FSH regulated endometrial atrophy by affecting the proliferation, autophagy and apoptosis of endometrial cells partly through activation of the transforming growth factor beta (TGFβ) pathway.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures

Several autophagy proteins contain an LC3‐interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B‐positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP‐ALFY‐LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR‐binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.     

Increased expression and functionality of the gap junction in peripheral blood lymphocytes is associated with hypertension-mediated inflammation in spontaneously hypertensive rats

Background

Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs).

Methods

The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A).

Results

The percentage of CD4⁺ T cells and the CD4⁺/CD8⁺ ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8⁺ and CD4⁺CD25⁺ T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes.

Conclusions

Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation.

     

Exercise training promotes expression of apelin and APJ of cardiovascular tissues in spontaneously hypertensive rats

Because apelin may play an important regulatory role in human cardiac dysfunction, we investigated alterations in cardiovascular content of apelin and its receptor, APJ, during hypertension and the effect of exercise training on the cardiovascular apelin/APJ system in hypertensive animals. Spontaneously hypertensive rats (SHRs) underwent swimming training consisting of 54 swimming sessions of 60 min each (6 days/week for 9 weeks). Systolic blood pressure (SBP) was verified weekly by tail-cuff plethysmography. Apelin levels in plasma and cardiovascular tissues were determined by radioimmunoassay. The level of apelin/APJ mRNA was determined by RT-PCR. SHRs showed severe hypertension and pathological cardiomegaly. The level of apelin immunoreactivity (apelin-ir) in plasma and ventricular and aortic tissues was lower, by 40%, 40% and 42% (all P<0.01), respectively, in SHRs than in control Wistar-Kyoto rats, and the mRNA level of apelin and APJ in myocardium and aorta was markedly decreased. Compared with sedentary SHRs, swimming-trained SHRs showed decreased SBP and elevated mRNA expression of apelin and APJ in cardiovascular tissues and elevated apelin-ir level in plasma, myocardium and aorta (all P<0.01). SBP and level of apelin-ir in plasma and cardiovascular tissues were negatively correlated. Long-term swimming training relieved the pathogenesis of hypertension and reversed the downregulation of the cardiovascular apelin/APJ system induced by hypertension, which suggests that the improving effect of exercise training on hypertension could be mediated by upregulating the cardiovascular apelin/APJ system.     

Connexin 31.1 degradation requires the Clathrin‐mediated autophagy in NSCLC cell H1299

Connexins have relative short half‐lives. Connexin 31.1 (Cx31.1) was newly reported to be down‐regulated in non‐small cell lung cancer cell lines, and displayed tumour‐suppressive properties. However, no reports describing how a cell regulates Cx31.1 level were found. In this study, Cx31.1 was suggested to be degraded through both ubiquitin–proteasome system (UPS) and autophagy. Blockage of UPS with MG‐132 increased Cx31.1 level, but could not inhibit the degradation of Cx31.1 completely. In H1299 cells stably expressing Cx31.1, Cx31.1 reduced when autophagy was induced through starvation or Brefeldin A treatment. Knockdown of autophagy‐related protein ATG5 could increase the cellular level of Cx31.1 both under normal growth condition and starvation‐induced autophagy. Colocalization of Cx31.1 and autophagy marker light chain 3 (LC3) was revealed by immunofluorescence analysis. Coimmunoprecipitation and immunofluorescence showed that Cx31.1 might interact with clathrin heavy chain which was newly reported to regulate autophagic lysosome reformation (ALR) and controls lysosome homoeostasis. When clathrin expression was knockdown by siRNA treatment, the level of Cx31.1 increased prominently both under normal growth condition and starvation‐induced autophagy. Under starvation‐induced autophagy, LC3‐II levels were slightly accumulated with siCla. treatment compared to that of siNC, which could be ascribed to that clathrin knockdown impaired the late stage of autophagy, ALR. Taken together, we found autophagy contributed to Cx31.1 degradation, and clathrin might be involved in the autophagy of Cx31.1.     

Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR‐204‐3p and inhibiting autophagy

This study was aimed at investigating the effects of lncRNA AK139328 on myocardial ischaemia/reperfusion injury (MIRI) in diabetic mice. Ischaemia/reperfusion (I/R) model was constructed in normal mice (NM) and diabetic mice (DM). Microarray analysis was utilized to identify lncRNA AK139328 overexpressed in DM after myocardial ischaemia/reperfusion (MI/R). RT‐qPCR assay was utilized to investigate the expressions of lncRNA AK139328 and miR‐204‐3p in cardiomyocyte and tissues. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening (FS) were obtained by transthoracic echocardiography. Haematoxylin‐eosin (HE) staining and Masson staining were utilized to detect the damage of myocardial tissues degradation of myocardial fibres and integrity of myocardial collagen fibres. Evans Blue/TTC staining was used to determine the myocardial infarct size. TUNEL staining was utilized to investigate cardiomyocyte apoptosis. The targeted relationship between lncRNA AK139328 and miR‐204‐3p was confirmed by dual‐luciferase reporter gene assay. MTT assay was used for analysis of cardiomyocyte proliferation. Western blot was utilized to investigate the expression of alpha smooth muscle actin (α‐SMA), Atg7, Atg5, LC3‐II/LC3‐I and p62 marking autophagy. Knockdown of lncRNA AK139328 relieved myocardial ischaemia/reperfusion injury in DM and inhibited cardiomyocyte autophagy as well as apoptosis of DM. LncRNA AK139328 modulated miR‐204‐3p directly. MiR‐204‐3p and knockdown of lncRNA AK139328 relieved hypoxia/reoxygenation injury via inhibiting cardiomyocyte autophagy. Silencing lncRNA AK139328 significantly increased miR‐204‐3p expression and inhibited cardiomyocyte autophagy, thereby attenuating MIRI in DM.     

Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β‐amyloid peptide

Alzheimer's disease (AD) is an aging‐related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP‐ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up‐regulated the lysoso‐mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3‐methylade‐nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ‐induced apoptosis.