scholarly journals Exogenous HGF Prevents Cardiomyocytes from Apoptosis after Hypoxia via Up-Regulating Cell Autophagy

2016 ◽  
Vol 38 (6) ◽  
pp. 2401-2413 ◽  
Author(s):  
Yunle Wang ◽  
Jiabao Liu ◽  
Zhiwen Tao ◽  
Peng Wu ◽  
Weili Cheng ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Protective Factor ◽  
Ventricular Myocytes ◽  
Early Stage ◽  
Neonatal Rat ◽  
Transmission Electron ◽  
Neonatal Rat Heart ◽  
Hoechst Staining ◽  
Hepatocyte Growth

Background: Hepatocyte growth factor (HGF) is widely known as a protective factor in ischemic myocardium, however HGF sensitive cellular mechanism remained ill-defined. Autophagy at early stage of hypoxia has been demonstrated to play a role in protecting myocardium both in vivo and vitro. We performed this study to investigate the association between the protective effect of HGF and autophagy. Methods: Ventricular myocytes were isolated from neonatal rat heart (NRVMs). We evaluated cardiomyocytes apoptosis by Hoechst staining and flow cytometry. Autophagy was assessed by transmission electron microscope and mRFP-GFP-LC3 adenovirus infection. Mitochondrial membrane potential was estimated by JC-1 staining. Western blotting and ELISA assay were used to quantify protein concentrations. Results: We found that autophagy in NRVMs increased at early stage after hypoxia and HGF release was consistent with the change of autophagy. Exogenous HGF enhanced autophagy and decreased apoptosis, while neutralizing HGF yielded opposite effects. Besides, inhibition of autophagy increased apoptosis of myocytes. Furthermore, exogenous HGF induced Parkin, the marker of mitochondrial autophagy, indicating increased clearance of injured mitochondria. Conclusions: Our results revealed a potential mechanism in which exogenous HGF prevented NRVMs from apoptosis after hypoxia. Upregulation of Parkin through administration of exogenous HGF may be a potential therapeutic strategy ptotecting myocytes during ischemia.

Abstract 631 ADAM12 Is An Important Regulator Of Myocyte Apoptosis Induced By β-adrenergic Receptor Stimulation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hang Xi ◽  
Khadija Rafiq ◽  
Marie Hanscom ◽  
Rachid Seqqat ◽  
Nikolay L Malinin ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Left Ventricular ◽  
Surface Proteins ◽  
Neonatal Rat ◽  
Human Heart Failure ◽  
Activation Mechanisms ◽  
Myocyte Apoptosis ◽  
Protease Deficient

ADAM (A Disintegrin And Metalloprotease)12 is a member of a family of cell surface proteins with protease and cell-binding activities. Recent work showed ADAM12 up-regulation in human heart failure. However, the activation mechanisms of ADAM12 in the heart are obscure. We hypothesized that β-adrenergic receptors (AR) stimulation regulates ADAM12 activation in neonatal rat ventricular myocytes (NRVMs) in-vitro and after injection of isoproterenol (ISO) in-vivo. Wistar rats received a single injection of ISO (5 mg/kg) and were sacrificed 6, 24 and 72 hrs later. In comparison with controls, left ventricular function was impaired in rats 24 hrs after ISO injection and started to improve at 72 hrs. The fraction of myocytes undergoing apoptosis peaked 24 hrs after ISO injection and declined thereafter. ADAM12 protein was reduced in hearts from ISO treated animals at 6 hrs, pointing to a possible increase in ADAM12 proteolytic activity. However, both ADAM12 expression and activation were significantly up-regulated at 24 and 72 hrs after ISO injection. We therefore assessed whether ADAM12 activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Acute stimulation with ISO (10 μM, 30 min to 3 hrs) induced accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease activity. However, chronic stimulation with ISO for 24 hrs and 48 hrs significantly increased both ADAM12 expression and secretion. This ISO-induced ADAM12 expression/activation was mediated through β 1 -AR stimulation and was dependent on intracellular calcium elevation and protein kinase C activation. Adenoviral expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks β-AR-induced myocyte apoptosis, while transduction of NRVMs with adenovirus harboring ADAM12 significantly increased myocyte apoptosis. These data suggest that ADAM12 is a regulator of myocyte apoptosis induced by β-AR in NRVMs and may play an important autocrine role in mediating the effects of β-AR on myocardial remodeling.

Ontogeny of prolyl endopeptidase, pyroglutamyl peptidase I, TRH, and its metabolites in rat pancreas

1992 ◽  
Vol 262 (6) ◽  
pp. E845-E850
Author(s):  
P. Salers ◽  
L. H. Ouafik ◽  
P. Giraud ◽  
J. Y. Maltese ◽  
A. Dutour ◽  
...  
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Early Stage ◽  
Prolyl Endopeptidase ◽  
Neonatal Rat ◽  
Rat Pancreas ◽  
Low Levels ◽  
Intact Rats

We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.

Abstract 16870: Dissociation of Mitochondrial HK-II Triggers Mitochondria Specific Autophagy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Shigeki Miyamoto ◽  
David J Roberts ◽  
Valerie P Tan-Sah
Keyword(s):  
Energy Production ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Hexokinase Ii ◽  
Survival Pathway ◽  
Mitochondrial Membrane Depolarization ◽  
Neonatal Rat Ventricular Myocytes

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.

Abstract 17473: GRK2 in the Mitochondria: Uncovering Novel Mechanisms in Heart Failure

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kimberly M Ferrero ◽  
Gizem Kayki Mutlu ◽  
Jessica M Pfleger ◽  
Douglas G Tilley ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dysfunction ◽  
Ventricular Myocytes ◽  
Atp Synthesis ◽  
Neonatal Rat ◽  
Negative Effects ◽  
Protein Kinase C Inhibitor ◽  
Neonatal Rat Ventricular Myocytes

Introduction: During heart failure, levels and activity of G protein-coupled receptor kinase 2 (GRK2) increase. GRK2 is canonically studied in the phosphorylation of GPCRs and β-adrenergic desensitization. Noncanonical activities of GRK2 are being uncovered, however. Our lab has recently discovered that in cardiac myocytes, GRK2 translocates to the mitochondria ( mtGRK2 ) following injury and is associated with negative effects on metabolism and cell survival. Hypothesis: GRK2 plays a role in regulating mitochondrial function following cardiac stress and contributes to HF pathogenesis in a novel manner, by interacting with a novel group of mitochondrial proteins involved in pro-death signaling, bioenergetics and substrate utilization. Methods: Mitochondrial translocation of GRK2 was validated with either protein kinase C inhibitor (chelerythine) administration or hypoxia/reoxygenation stress in primary neonatal rat ventricular myocytes or a cardiac-like cell line. Immunoprecipitation of the GRK2 interactome basally and under stress conditions was conducted endogenously in vitro, in vivo , and with purified recombinant GRK2 peptides. Proteins were separated via SDS-PAGE and potential binding partners were identified by mass spectroscopy (LCMS) and proteomics analysis conducted with Ingenuity Pathway (IPA; Qiagen) software to determine which partners in the GRK2 interactome were potentially involved in mitochondrial dysfunction. Results: Subunits of Complexes I, II, IV and V of the electron transport chain were identified as potential mtGRK2 interacting partners. Several mtGRK2-ETC interactions were increased following oxidative stress-induced translocation of GRK2. Finally, mtGRK2 appears to phosphorylate some of the interactome partners identified in mitochondrial dysfunction. Conclusions: The phosphorylation of subunits of the ATP synthesis machinery by mtGRK2, or other mechanisms of interaction between these proteins, may be regulating some of the phenotypic effects of HF previously observed by our lab, such as increased ROS production and reduced fatty acid metabolism. Further research is essential to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death in failing hearts.

scholarly journals Ultrastructure and intercellular contact-mediated communication in cultured human early stage follicles exposed to mTORC1 inhibitor

2019 ◽  
Vol 25 (11) ◽  
pp. 706-716 ◽  
Author(s):  
J Grosbois ◽  
M Vermeersch ◽  
M Devos ◽  
H J Clarke ◽  
I Demeestere
Keyword(s):  
Early Stage ◽  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Control Group ◽  
Mediated Communication ◽  
Transmission Electron ◽  
Primordial Follicle Activation ◽  
Intercellular Communications ◽  
Follicle Activation

Abstract The reproductive lifespan of a woman is determined by the gradual recruitment of quiescent follicles into the growing pool. In humans, ovarian tissue removal from its in vivo environment induces spontaneous activation of resting follicles. Similarly, pharmacological activation of the PI3K/Akt pathway leads to accelerated follicle recruitment, but has been associated with follicular damage. Recent findings demonstrate that everolimus (EVE), an mTORC1 inhibitor, limits primordial follicle activation. However, its potential benefit regarding growing follicle integrity remains unexplored. Ovarian cortical fragments were exposed to ± EVE for 24 h and cultured for an additional 5 days. After 0, 1 and 6 days of culture, fragments were either processed for ultrastructural analysis or subjected to follicular isolation for gene expression and immunofluorescence assessments. Data from transmission electron microscopy showed that growing follicles displayed similar ultrastructural features irrespective of the conditions and maintained close contacts between germinal and stromal compartments. Establishment of intra-follicular communication was confirmed by detection of a gap junction component, Cx43, in both groups throughout culture, whereas transzonal projections, which physically link granulosa cells to oocyte, formed later in EVE-treated follicles. Importantly, levels of GJA1 mRNA, encoding for the Cx43 protein, significantly increased from Day 0 to Day 1 in the EVE group, but not in the control group. Given that EVE-treated follicles were smaller than controls, these findings suggest that EVE might facilitate the establishment of appropriate intercellular communications without impairing follicle ultrastructure. Therefore, mTORC1 inhibitors might represent an attractive tool to delay the culture-induced primordial follicle activation while maintaining follicles in a functionally integrated state.

scholarly journals CMTM7 as a novel molecule of ATG14L-Beclin1-VPS34 complex enhances autophagy by Rab5 to regulate tumorigenicity

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Baocai Liu ◽  
Yinliang Lu ◽  
Tingting Zhang ◽  
Xinyue Yu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Early Stage ◽  
Xenograft Model ◽  
Vital Role ◽  
Autophagic Flux ◽  
In Vivo Experiments ◽  
Transmission Electron ◽  
Autophagy Induction

Abstract Background CMTM7 is a tumor suppressor that positively regulates EGFR degradation by promoting Rab5 activation, and plays a vital role in tumor progression. Rab5 forms complexes with Beclin1 and VPS34, and acts in the early stage of autophagy. However, the affects of CMTM7 on autophagy and its mechanism are still unclear. Methods The effect of CMTM7 on autophagy induction was confirmed by western blotting, confocal microscopy and transmission electron microscopy. Co-immunoprecipitation was used to analyse the interaction of CMTM7 with autophagy initiation complex and Rab5. The xenograft model in nude mice was used to elucidate the function of CMTM7 in tumorigenicity and autophagy in vivo. Results In this study, we first demonstrated that CMTM7 facilitated the initiation of autophagosome formation, which consequently promoted the subsequent multistage process of autophagic flux, i.e. from autophagosome assembly till autolysosome formation and degradation. Confocal and co-immunoprecipitation showed that CMTM7 interacted with Rab5, VPS34, Beclin1, and ATG14L, but not with ULK1, UVRAG and LC3B. CMTM7 also increased the activity of ATG14L-linked VPS34 complex and its association with Rab5. Both in vitro and in vivo experiments demonstrated that knockdown of CMTM7 enhanced tumor growth by impairing autophagy. Conclusion These findings highlighted the role of CMTM7 in the regulation of autophagy and tumorigenicity, revealing it as a novel molecule that is associated with the interaction of Rab5 and ATG14L-Beclin1-VPS34 complex.

scholarly journals RIP1/RIP3/MLKL-mediated necroptosis contributes to vinblastine-induced myocardial damage

2020 ◽  
Author(s):  
Huiling Zhou ◽  
Lijun Liu ◽  
Xiaolong Ma ◽  
Jian Wang ◽  
Jinfu Yang ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Cell Damage ◽  
Animal Experiments ◽  
Myocardial Damage ◽  
Myocardial Cell ◽  
Neonatal Rat ◽  
H9c2 Cells ◽  
Interacting Protein

AbstractVinblastine (VBL) has been considered as a first-line anti-tumor drug for many years. However, vinblastine-caused myocardial damage has been continually reported. The underlying molecular mechanism of the myocardial damage remains unknown. Here, we show that vinblastine induces myocardial damage and necroptosis is involved in the vinblastine-induced myocardial damage both in vitro and in vivo. The results of WST-8 and flow cytometry analysis show that vinblastine causes damage to H9c2 cells, and the results of animal experiments show that vinblastine causes myocardial cell damage. The necrosome components, receptor-interacting protein 1 (RIP1) receptor-interacting protein 3 (RIP3), are significantly increased in vinblastine-treated H9c2 cells, primary neonatal rat ventricular myocytes and rat heart tissues. And the downstream substrate of RIP3, mixed lineage kinase domain like protein (MLKL) was also increased. Pre-treatment with necroptosis inhibitors partially inhibits the necrosome components and MLKL levels and alleviates vinblastine-induced myocardial injury both in vitro and in vivo. This study indicates that necroptosis participated in vinblastine-evoked myocardial cell death partially, which would be a potential target for relieving the chemotherapy-related myocardial damage.

scholarly journals NIR-II emissive AIEgen photosensitizers enable ultrasensitive imaging-guided surgery and phototherapy to fully inhibit orthotopic hepatic tumors

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ruizhen Jia ◽  
Han Xu ◽  
Chenlu Wang ◽  
Lichao Su ◽  
Jinpeng Jing ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Near Infrared ◽  
Liver Tumors ◽  
Early Stage ◽  
Oxygen Species ◽  
Hepatic Tumors ◽  
Primary Liver Tumors ◽  
Guided Surgery

AbstractAccurate diagnosis and effective treatment of primary liver tumors are of great significance, and optical imaging has been widely employed in clinical imaging-guided surgery for liver tumors. The second near-infrared window (NIR-II) emissive AIEgen photosensitizers have attracted a lot of attention with higher-resolution bioimaging and deeper penetration. NIR-II aggregation-induced emission-based luminogen (AIEgen) photosensitizers have better phototherapeutic effects and accuracy of the image-guided surgery/phototherapy. Herein, an NIR-II AIEgen phototheranostic dot was proposed for NIR-II imaging-guided resection surgery and phototherapy for orthotopic hepatic tumors. Compared with indocyanine green (ICG), the AIEgen dots showed bright and sharp NIR-II emission at 1250 nm, which extended to 1600 nm with high photostability. Moreover, the AIEgen dots efficiently generated reactive oxygen species (ROS) for photodynamic therapy. Investigations of orthotopic liver tumors in vitro and in vivo demonstrated that AIEgen dots could be employed both for imaging-guided tumor surgery of early-stage tumors and for ‘downstaging’ intention to reduce the size. Moreover, the therapeutic strategy induced complete inhibition of orthotopic tumors without recurrence and with few side effects. Graphical Abstract

Abstract 4626: Optimal Dose of Adv-hHCN4 Creates Stable and Long-lasting Biopacing Activity in Cultured Ventricular Cardiomyocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yong-Fu Xiao ◽  
Alena Nikolskaya ◽  
Lepeng Zeng ◽  
Xiaohong Qiu ◽  
Deborah A. Jaye ◽  
...  
Keyword(s):  
Channel Blocker ◽  
Ventricular Myocytes ◽  
Optimal Dose ◽  
Neonatal Rat ◽  
Normal Morphology ◽  
Rat Ventricular Myocytes ◽  
Beating Rate ◽  
Neonatal Rat Ventricular Myocytes

Purpose: Hyperpolarization-activated cyclic nucleotide-gated (HCN) genes have been successfully used as a strategy for recreating cardiac biological pacemakers in animal models. However, optimal dose of HCN and toxicity from HCN overexpression have not been investigated. Therefore, we assessed the effects of various titers of adenoviral human HCN4-GFP vector (Adv-hHCN4) on cardiomyocytes. Methods: Neonatal rat ventricular myocytes (NRVMs) were isolated, selected and cultured on microelectrode arrays to assess their automaticities. Morphology and apoptosis with and without HCN or Ca 2+ channel inhibitor were also assessed. Results: Beating rates significantly increased in NRVMs after hHCN4 infection (Fig. 1 ). For example, the rates were gradually increased to 235±11 beat/min on day 7 after hHCN4 infection with 1×10 5 PFU/array. In contrast, control cells showed low rates. NRVMs with ≥10 6 PFU/array Adv-hHCN4 reached faster rates early and subsequently stopped beating (Fig. 1 ). In addition, myocytes with ≥10 6 PFU/array Adv-hHCN4 underwent significant apoptosis (>50%) which potentially resulted from hHCN4 overexpression and was blocked by the HCN channel blocker Cs + (1 mM), but not by the Ca 2+ channel inhibitor nifedipine. In addition, myocytes infected with ≥10 6 PFU/array Adv-GFP maintained normal morphology and rate. Our data demonstrate that hHCN4 transfer significantly and dose-dependently increased beating rates of NRVMs. However, overexpression of HCN could cause apoptosis. Therefore, an optimal dose of HCN gene is important for reducing toxicity and creating stable and long-lasting biopacing activity in cardiomyocytes in vitro, and probably also in vivo. Figure 1. Effects of hHCN4 infection on automaticities of neonatal rat ventricular myocytes. Each data point represents an averaged beating rate (mean ± SE) from 8 to 10 arrays. Various titers (1×10 5 to 1×10 7 PFU/500,000 cells per array) of Adv-Hhcn4 (expect control) were added to the arrays after measurements on day 0 (see the arrow)

scholarly journals Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes

1997 ◽  
Vol 325 (3) ◽  
pp. 801-810 ◽  
Author(s):  
Angela CLERK ◽  
Peter H. SUGDEN
Keyword(s):  
Transcription Factors ◽  
Okadaic Acid ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Western Blots ◽  
Hyperosmotic Shock ◽  
Nuclear Extracts

Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro,extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.

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