Abstract MP231: Long Non-coding Rna Camirt Plays A Sentinel Role In Aging-related Heart Failure Via Interaction With Phb2 To Modulate Mitophagy Signaling In The Heart

Background: Mitochondrial dysfunction is an important risk factor for heart failure in elderly people. Mitophagy, a physiological process that controls the removal of damaged mitochondria, is compromised in aging or failing hearts. In this study, we examined the physiological role of a cardiac-specific lncRNA Camirt that can potentially modulate mitophagy in the heart. Methods and Results: RNA-seq analysis and RT-PCR reveal a lncRNA is highly expressed in both mouse and human hearts, with undetectable levels in other vital organs. Furthermore, Real time qPCR was used to examine the expression of lncRNA in different animal models and in human hearts, which results showed that the expression of this lncRNA is decreased in aging mouse and human hearts, and failing mouse hearts induced by isoproterenol and doxorubicin. RNA pull-down and RNA-binding protein immunoprecipitation assays identify prohibitin-2 (Phb2), a known mitophagy receptor, as a binding partner for this lncRNA. Thus, we name this novel lncRNA as a cardiac-specific mitophagy-associated RNA transcript (Camirt). Camirt conditional (flox) knockout mice were created via CRISPR /Cas9 technology, and subjected to the longitudinal echocardiographic and survival studies. Mice with cardiac specific deletion of Camirt (Camirt-cKO) display progressive heart failure and die within 12 month after birth. RNA sequencing and gene ontology analysis revealed that genes involved in mitophagy signaling were significantly altered in the Camirt-cKO hearts compared with the littermate wild type mice. Transmission electron microscopy were used to examine the mitochondrial morphology in mouse hearts, and reveal excessive accumulation of mitolysosomes in cardiomyocytes derived from the Camirt-cKO mice. In vitro study with Annexin-V/PI staining showed an increased number of live cells and decreased number of apoptotic cells in cultured neonatal cardiomyocytes with overexpression of Camirt following oxidative stress induced by treatment with H2O2. Increased autophagy (or mitophagy) activity was observed in HL-1 cells with stable overexpression of Camirt and in the presence of chloroquine (an inhibitor for the lysosome degradation). While reduced Camirt expression via shRNA knock-down leads to compromised mitophagy activity in HL-1 cells. Further biochemical studies support the function of Camirt/Phb2 in maintenance of mitochondria function and mitophagy signaling under stress conditions. Conclusion: Overall, our results suggested that Camirt plays a sentinel role in aging-related heart failure via interaction with Phb2 to modulate mitophagy signaling in the heart. Future studies will focus on elucidating the in vivo role and mechanisms of Camirt in modulation of mitophagy under natural aging or stress-induced pathologic conditions using the loss- or gain-of-function of Camirt mouse models.

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Increased Bone Mass in Mice Lacking the Adipokine Apelin

Endocrinology ◽

10.1210/en.2012-2034 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2069-2080 ◽

Cited By ~ 22

Author(s):

Lalita Wattanachanya ◽

Wei-Dar Lu ◽

Ramendra K. Kundu ◽

Liping Wang ◽

Marcia J. Abbott ◽

...

Keyword(s):

Bone Mass ◽

Bone Cells ◽

Physiological Role ◽

In Vitro Study ◽

Maximum Effect ◽

Dependent Manner ◽

Primary Mouse ◽

Skeletal Homeostasis

Abstract Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

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Sympathoexcitation in chronic heart failure: Ang II induced inhibition of voltage‐gated K + channel, an in vivo and in vitro study

The FASEB Journal ◽

10.1096/fasebj.20.5.a1202-c ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

LIE GAO ◽

Wei Wang ◽

Ethan Mann ◽

Marcus Finch ◽

Yulong Li ◽

...

Keyword(s):

Heart Failure ◽

Chronic Heart Failure ◽

In Vitro Study ◽

Vitro Study ◽

K Channel ◽

Ang Ii ◽

Voltage Gated

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In Vitro and In Vivo Antitumor Activity of Silver Nanoparticles on B16 Melanoma

NANO ◽

10.1142/s1793292020501635 ◽

2020 ◽

Vol 15 (12) ◽

pp. 2050163

Author(s):

Hongkun Gao ◽

Ping Fan ◽

Qizhen Xu ◽

Yiting Li ◽

Jianxin Wang ◽

...

Keyword(s):

Silver Nanoparticles ◽

Antitumor Activity ◽

Antimicrobial Agents ◽

Malignant Tumors ◽

Annexin V ◽

In Vitro Study ◽

B16 Melanoma ◽

Potential Strategy

Melanoma, one of the most malignant tumors, is difficult to treat due to its high drug resistance. Silver nanoparticles (AgNPs) are widely used as antimicrobial agents in biomedical fields. In this study, the spherical AgNPs with average sizes of 5[Formula: see text]nm were prepared using a dopamine reduction method. The in vitro study shows that AgNPs with the concentrations of 0.5[Formula: see text][Formula: see text]g/mL and 1[Formula: see text][Formula: see text]g/mL exhibit good biocompatibility to 3T3L1 fibroblast cells. AgNPs with the same concentrations significantly inhibited the growth of B16 melanoma cells. In culture with B16 cells, AgNPs induced intracellular oxidative stress by generating the reactive oxygen species and reducing the superoxide dismutase, which further reduces the mitochondrial membrane potential. Moreover, the damage in mitochondria could activate mitochondrion-mediated cell apoptosis. The B16 cells apoptosis was analyzed by FITC-Annexin V/propidium iodide double staining assay, which confirms that AgNPs caused the abundance of apoptotic cells in different stages. Thus, AgNPs displayed the antitumor activity in vitro. Then, the therapeutic efficacy in vivo was evaluated in mice-bearing B16 melanoma tumors. The obtained results show the antitumor ability of AgNPs and provide a potential strategy for cancer treatment.

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Detailed Analysis of Apoptotic Responses to Cytokine Stimulation in B-Cell Chronic Lymphocytic Leukaemia.

Blood ◽

10.1182/blood.v104.11.4773.4773 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4773-4773

Author(s):

Andrew P. Jewell ◽

Wendy Smith ◽

Rob van Schie ◽

Nina Porakishvili ◽

Peter M. Lydyard

Keyword(s):

B Cell ◽

Interferon Gamma ◽

Annexin V ◽

Lymphocytic Leukemia ◽

Live Cells ◽

Tnf Alpha ◽

Gm Csf ◽

Cytokine Stimulation

Abstract Introduction B cell Chronic lymphocytic leukemia (BCLL) is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo, but are susceptible to apoptosis when incubated in vitro. A number of cytokines have been reported to protect against apoptosis in vitro, but it is not clear whether the effects of these cytokines are mediated in similar ways. We have therefore studied the regulation of apoptosis in B-CLL cells in vitro, in response to various cytokines. Methods B-CLL cells were purified and incubated with the following agents; Interferon-gamma, IL-4, IL-10, IL-13, TNF-alpha and GM-CSF, each at 3 different concentrations. Apoptosis was measured after 70 hours by Annexin/PI staining and analysed for early apoptosis (Annexin V positive), late apoptotic (Annexin V and PI positive), and necrotic (PI positive). Results The percentage of live cells in unstimulated B-CLL cells was 42.4±2.3%. Co-incubation of B-CLL cells with IL-4 at 100ng/ml increased the percentage of live cells to 63.2±8.4%, while in the presence of interferon-gamma the percentage of live cells was 48.7±5.9%. In contrast, co-incubation with TNF-alpha reduced the percentage of live cells to 30.8±6.8%. IL-10, IL-13 and GM-CSF had no effect on B-CLL cells survival at any of the concentrations used.. No significant differences in the pattern of early/late apoptosis or necrosis were seen between the different cytokines used. Figure Figure

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A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00079.2004 ◽

2004 ◽

Vol 287 (3) ◽

pp. H1096-H1103 ◽

Cited By ~ 36

Author(s):

Alexander Yang ◽

Dimitry Sonin ◽

Larry Jones ◽

William H. Barry ◽

Bruce T. Liang

Keyword(s):

Heart Failure ◽

Purinergic Receptor ◽

Weight Ratio ◽

Physiological Role ◽

Premature Death ◽

Left Ventricular ◽

Developed Pressure

The P2X4 purinergic receptor (P2X4R) is a ligand-gated ion channel. Its activation by extracellular ATP results in Ca2+ influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 ± 91 days for the binary CSQ/P2X4R mouse, n = 35) of the CSQ mouse (71.3 ± 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored β-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and ±dP/d t. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure.

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Hispidulin Attenuates Cardiac Hypertrophy by Improving Mitochondrial Dysfunction

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.582890 ◽

2020 ◽

Vol 7 ◽

Author(s):

Yan Wang ◽

Zengshuo Xie ◽

Nan Jiang ◽

Zexuan Wu ◽

Ruicong Xue ◽

...

Keyword(s):

Oxidative Stress ◽

Heart Failure ◽

Cardiac Hypertrophy ◽

Mitochondrial Function ◽

Specific Inhibitor ◽

Cardiomyocyte Hypertrophy ◽

Mitochondrial Morphology ◽

Protective Effects

Cardiac hypertrophy is a pathophysiological response to harmful stimuli. The continued presence of cardiac hypertrophy will ultimately develop into heart failure. The mitochondrion is the primary organelle of energy production, and its dysfunction plays a crucial role in the progressive development of heart failure from cardiac hypertrophy. Hispidulin, a natural flavonoid, has been substantiated to improve energy metabolism and inhibit oxidative stress. However, how hispidulin regulates cardiac hypertrophy and its underlying mechanism remains unknown. We found that hispidulin significantly inhibited pressure overload-induced cardiac hypertrophy and improved cardiac function in vivo and blocked phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. We further proved that hispidulin remarkably improved mitochondrial function, manifested by increased electron transport chain (ETC) subunits expression, elevated ATP production, increased oxygen consumption rates (OCR), normalized mitochondrial morphology, and reduced oxidative stress. Furthermore, we discovered that Sirt1, a well-recognized regulator of mitochondrial function, might be a target of hispidulin, as evidenced by its upregulation after hispidulin treatment. Cotreatment with EX527 (a Sirt1-specific inhibitor) and hispidulin nearly completely abolished the antihypertrophic and protective effects of hispidulin on mitochondrial function, providing further evidence that Sirt1 could be the pivotal downstream effector of hispidulin in regulating cardiac hypertrophy.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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