Abstract 319: Role of Phosphodiesterases 4B and 4D in cAMP Signal Compartmentation Around Calcium Handling Proteins

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Axel E Kraft ◽  
Viacheslav O Nikolaev ◽  
Marco Conti
Keyword(s):  
Knockout Mice ◽  
Heart Function ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Specific Effect ◽  
Resonance Energy ◽  
Direct Analysis ◽  
Calcium Handling ◽  
Camp Signaling

Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations in activity of these enzymes in human hearts have been shown to result in arrhythmia and heart failure. The aim of this project was to systematically investigate specific roles of PDE4B and PDE4D in regulating cAMP dynamics in three distinct subcellular microdomains formed around Ca 2+ handling proteins, such as L-type calcium channels (LTCCs), sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) and ryanodine receptors (RyRs), to understand their impact on heart function and disease. Transgenic mice expressing three different Förster resonance energy transfer (FRET) based cAMP biosensors targeted to caveolin rich plasma membrane, SERCA and RyR microdomains, were crossed with PDE4B and PDE4D knockout mice. Using FRET imaging in ventricular cardiomyocytes freshly isolated from wildtype and knockout mice, direct analysis of the specific effect of both PDE subfamilies in these microdomains could be performed by measuring the kinetics of local cAMP degradation. Our results indicate that the cAMP kinetics around the LTCC microdomain is critically regulated by PDE4B and PDE4D. So far was it has been known that the isoform that is associated with the RyR microdomain belongs to the PDE4D family, however we found PDE4B to be involved in regulating the cAMP signaling in this microdomain. PDE4D deletion also revealed the critical role of this subfamily for the control of cAMP dynamics in the SERCA microdomain of adult mouse cardiomyocytes. Basal levels of cAMP were elevated when PDE4B was absent from any of the PDE4B-regulated microdomain, whereas no such alterations were detected for PDE4D knockout cells. These data demonstrate that all three microdomains are differentially regulated by PDEs. Even within one organelle such as sarcoplasmic reticulum, we could show the existence of at least two distinct cAMP microdomains, i.e. around RyR and SERCA which are preferentially controlled by PDE4B and PDE4D, respectively. In the future, we aim to systematically analyze biochemical composition of the three microdomains, their distinct roles in cardiac function and disease as well as ways of their pharmacological modulation.

Overexpression of Pygo2 Increases Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Cardiomyocyte-like Cells

2020 ◽  
Vol 20 (4) ◽  
pp. 318-324 ◽  
Author(s):  
Lei Yang ◽  
Shuoji Zhu ◽  
Yongqing Li ◽  
Jian Zhuang ◽  
Jimei Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Heart Function ◽  
Critical Role ◽  
Human Umbilical Cord ◽  
Stem Cell Markers ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr

Background: Our previous studies have shown that Pygo (Pygopus) in Drosophila plays a critical role in adult heart function that is likely conserved in mammals. However, its role in the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into cardiomyocytes remains unknown. Objective: To investigate the role of pygo2 in the differentiation of hUC-MSCs into cardiomyocytes. Methods: Third passage hUC-MSCs were divided into two groups: a p+ group infected with the GV492-pygo2 virus and a p− group infected with the GV492 virus. After infection and 3 or 21 days of incubation, Quantitative real-time PCR (qRT-PCR) was performed to detect pluripotency markers, including OCT-4 and SOX2. Nkx2.5, Gata-4 and cTnT were detected by immunofluorescence at 7, 14 and 21 days post-infection, respectively. Expression of cardiac-related genes—including Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin—were analyzed by qRT-PCR following transfection with the virus at one, two and three weeks. Results : After three days of incubation, there were no significant changes in the expression of the pluripotency stem cell markers OCT-4 and SOX2 in the p+ group hUC-MSCs relative to controls (OCT-4: 1.03 ± 0.096 VS 1, P > 0.05, SOX2: 1.071 ± 0.189 VS 1, P > 0.05); however, after 21 days, significant decreases were observed (OCT-4: 0.164 ± 0.098 VS 1, P < 0.01, SOX2: 0.209 ± 0.109 VS 1, P < 0.001). Seven days following incubation, expression of mesoderm specialisation markers, such as Nkx2.5, Gata-4, MEF2c and KDR, were increased; at 14 days following incubation, expression of cardiac genes, such as Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin, were significantly upregulated in the p+ group relative to the p− group (P < 0.05). Taken together, these findings suggest that overexpression of pygo2 results in more hUCMSCs gradually differentiating into cardiomyocyte-like cells. Conclusion: We are the first to show that overexpression of pygo2 significantly enhances the expression of cardiac-genic genes, including Nkx2.5 and Gata-4, and promotes the differentiation of hUC-MSCs into cardiomyocyte-like cells.

scholarly journals Critical Role of PDE4D in β2-Adrenoceptor-dependent cAMP Signaling in Mouse Embryonic Fibroblasts

2008 ◽  
Vol 283 (33) ◽  
pp. 22430-22442 ◽  
Author(s):  
Matthew D. Bruss ◽  
Wito Richter ◽  
Kathleen Horner ◽  
S.-L. Catherine Jin ◽  
Marco Conti
Keyword(s):  
Critical Role ◽  
Camp Signaling ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals Regulation of the cognitive aging process by the transcriptional repressor RP58

2021 ◽  
Author(s):  
Tomoko Tanaka ◽  
Shinobu Hirai ◽  
Hiroyuki Manabe ◽  
Kentaro Endo ◽  
Hiroko Shimbo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Knockout Mice ◽  
Cognitive Aging ◽  
Critical Role ◽  
Harmful Effect ◽  
Transcriptional Repressor ◽  
Repair Pathway ◽  
Wild Type

Aging involves a decline in physiology which is a natural event in all living organisms. An accumulation of DNA damage contributes to the progression of aging. DNA is continually damaged by exogenous sources and endogenous sources. If the DNA repair pathway operates normally, DNA damage is not life threatening. However, impairments of the DNA repair pathway may result in an accumulation of DNA damage, which has a harmful effect on health and causes an onset of pathology. RP58, a zinc-finger transcriptional repressor, plays a critical role in cerebral cortex formation. Recently, it has been reported that the expression level of RP58 decreases in the aged human cortex. Furthermore, the role of RP58 in DNA damage is inferred by the involvement of DNMT3, which acts as a co-repressor for RP58, in DNA damage. Therefore, RP58 may play a crucial role in the DNA damage associated with aging. In the present study, we investigated the role of RP58 in aging. We used RP58 hetero-knockout and wild-type mice in adolescence, adulthood, or old age. We performed immunohistochemistry to determine whether microglia and DNA damage markers responded to the decline in RP58 levels. Furthermore, we performed an object location test to measure cognitive function, which decline with age. We found that the wild-type mice showed an increase in single-stranded DNA and gamma-H2AX foci. These results indicate an increase in DNA damage or dysfunction of DNA repair mechanisms in the hippocampus as age-related changes. Furthermore, we found that, with advancing age, both the wild-type and hetero-knockout mice showed an impairment of spatial memory for the object and increase in reactive microglia in the hippocampus. However, the RP58 hetero-knockout mice showed these symptoms earlier than the wild-type mice did. These results suggest that a decline in RP58 level may lead to the progression of aging.

Abstract P080: Bestrophin: A Potential Mediator of Cardiac Calcium Handling

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Fiona C Britton ◽  
Steeven John ◽  
Kate E O'Driscoll ◽  
Martha Baring
Keyword(s):  
Chloride Channels ◽  
Calcium Release ◽  
Heart Function ◽  
Critical Role ◽  
Calcium Handling ◽  
Positive Interactions ◽  
Binding Motif ◽  
Mouse Heart ◽  
Charge Movement ◽  
Interaction Sites

Bestrophins are a family of transmembrane proteins expressed in heart. We reported that Bestrophin 1 and 3 cloned from mouse heart function as calcium-activated chloride channels. We hypothesized that bestrophin channels may function in a macromolecule complex similar to other cardiac ion channels. To identify proteins that bind Bestrophin 3 channel, we screened a mouse heart cDNA library using 4 different cytosolic cDNA fragments as bait. Yeast two-hybrid assays were performed with the GAL4 system. Using bait consisting of C-terminus amino acid residues 386 to 669 of the Best3 channel (Best3-C2), we obtained positive interactions with the histidine-rich calcium binding protein (hrc). Hrc is a component of the SR complex involved in calcium handling in the heart. Eleven independent clones represented a fragment encoding the cysteine rich domain of hrc previously shown to interact with triadin. To further map the interaction sites between Best3-C2 and hrc, we subdivided this region into three fragments and tested these baits for interaction with hrc. Two Best3 fragments (residues 541–600 and residues 601–669) displayed strong interaction with hrc, while Best3 bait (residues 386–540) failed to interact. This region of Best3 contains a KEKE protein binding motif. In a similar manner C-terminal Best 1 fragments were confirmed to also interact with hrc. In this study we report that Bestrophin channels interact with hrc, a cardiac calcium handling protein. Calcium-activated chloride channels play a critical role in excitation-contraction coupling in the SR by balancing charge movement during calcium release and reuptake. Recent evidence confirms Bestrophin 1 is located in the ER membrane in association with Stim1 and facilitates calcium cycling. Future in vivo investigations will examine the role of bestrophins as potential mediators of calcium handling in heart.

scholarly journals Critical role of spectrin in hearing development and deafness

2019 ◽  
Vol 5 (4) ◽  
pp. eaav7803 ◽  
Author(s):  
Yan Liu ◽  
Jieyu Qi ◽  
Xin Chen ◽  
Mingliang Tang ◽  
Cenfeng Chu ◽  
...  
Keyword(s):  
Hair Cells ◽  
Knockout Mice ◽  
Crucial Role ◽  
Structural Organization ◽  
Critical Role ◽  
Super Resolution ◽  
Mechanical Vibrations ◽  
Hearing Ability ◽  
Profound Deafness

Inner ear hair cells (HCs) detect sound through the deflection of mechanosensory stereocilia. Stereocilia are inserted into the cuticular plate of HCs by parallel actin rootlets, where they convert sound-induced mechanical vibrations into electrical signals. The molecules that support these rootlets and enable them to withstand constant mechanical stresses underpin our ability to hear. However, the structures of these molecules have remained unknown. We hypothesized that αII- and βII-spectrin subunits fulfill this role, and investigated their structural organization in rodent HCs. Using super-resolution fluorescence imaging, we found that spectrin formed ring-like structures around the base of stereocilia rootlets. These spectrin rings were associated with the hearing ability of mice. Further, HC-specific, βII-spectrin knockout mice displayed profound deafness. Overall, our work has identified and characterized structures of spectrin that play a crucial role in mammalian hearing development.

scholarly journals Disruption of CXCR6 Ameliorates Kidney Inflammation and Fibrosis in Deoxycorticosterone Acetate/Salt Hypertension

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuanbo Wu ◽  
Changlong An ◽  
Xiaogao Jin ◽  
Zhaoyong Hu ◽  
Yanlin Wang
Keyword(s):  
Knockout Mice ◽  
Critical Role ◽  
Kidney Injury ◽  
Wild Type ◽  
Salt Treatment ◽  
Hypertensive Nephropathy ◽  
Deoxycorticosterone Acetate ◽  
Kidney Fibrosis ◽  
Salt Hypertension

AbstractCirculating cells have a pathogenic role in the development of hypertensive nephropathy. However, how these cells infiltrate into the kidney are not fully elucidated. In this study, we investigated the role of CXCR6 in deoxycorticosterone acetate (DOCA)/salt-induced inflammation and fibrosis of the kidney. Following uninephrectomy, wild-type and CXCR6 knockout mice were treated with DOCA/salt for 3 weeks. Blood pressure was similar between wild-type and CXCR6 knockout mice at baseline and after treatment with DOCA/salt. Wild-type mice develop significant kidney injury, proteinuria, and kidney fibrosis after three weeks of DOCA/salt treatment. CXCR6 deficiency ameliorated kidney injury, proteinuria, and kidney fibrosis following treatment with DOCA/salt. Moreover, CXCR6 deficiency inhibited accumulation of bone marrow–derived fibroblasts and myofibroblasts in the kidney following treatment with DOCA/salt. Furthermore, CXCR6 deficiency markedly reduced the number of macrophages and T cells in the kidney after DOCA/salt treatment. In summary, our results identify a critical role of CXCR6 in the development of inflammation and fibrosis of the kidney in salt-sensitive hypertension.

scholarly journals SNAP-23 regulates phagosome formation and maturation in macrophages

2012 ◽  
Vol 23 (24) ◽  
pp. 4849-4863 ◽  
Author(s):  
Chiye Sakurai ◽  
Hitoshi Hashimoto ◽  
Hideki Nakanishi ◽  
Seisuke Arai ◽  
Yoh Wada ◽  
...  
Keyword(s):  
Critical Role ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Nadph Oxidase Complex ◽  
Uptake Activity ◽  
Fret Efficiency ◽  
Precise Role

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.

scholarly journals Increased Susceptibility to Isoproterenol-Induced Cardiac Hypertrophy and Impaired Weight Gain in Mice Lacking the Histidine-Rich Calcium-Binding Protein

2006 ◽  
Vol 26 (24) ◽  
pp. 9315-9326 ◽  
Author(s):  
Eric J. Jaehnig ◽  
Analeah B. Heidt ◽  
Stephanie B. Greene ◽  
Ivo Cornelissen ◽  
Brian L. Black
Keyword(s):  
Weight Gain ◽  
Cardiac Hypertrophy ◽  
Knockout Mice ◽  
Calcium Binding ◽  
Calcium Release ◽  
Binding Protein ◽  
Critical Role ◽  
Calcium Binding Protein ◽  
Calcium Handling

ABSTRACT The sarcoplasmic reticulum (SR) plays a critical role in excitation-contraction coupling by regulating the cytoplasmic calcium concentration of striated muscle. The histidine-rich calcium-binding protein (HRCBP) is expressed in the junctional SR, the site of calcium release from the SR. HRCBP is expressed exclusively in muscle tissues and binds calcium with low affinity and high capacity. In addition, HRCBP interacts with triadin, a protein associated with the ryanodine receptor and thought to be involved in calcium release. Its calcium binding properties, localization to the SR, and interaction with triadin suggest that HRCBP is involved in calcium handling by the SR. To determine the function of HRCBP in vivo, we inactivated HRC, the gene encoding HRCBP, in mice. HRC knockout mice exhibited impaired weight gain beginning at 11 months of age, which was marked by reduced skeletal muscle and fat mass, and triadin protein expression was upregulated in the heart of HRC knockout mice. In addition, HRC null mice displayed a significantly exaggerated response to the induction of cardiac hypertrophy by isoproterenol compared to their wild-type littermates. The exaggerated response of HRC knockout mice to the induction of cardiac hypertrophy is consistent with a regulatory role for HRCBP in calcium handling in vivo and suggests that mutations in HRC, in combination with other genetic or environmental factors, might contribute to pathological hypertrophy and heart failure.

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

2006 ◽  
Vol 290 (2) ◽  
pp. C539-C553 ◽  
Author(s):  
Jingsong Zhou ◽  
Jianxun Yi ◽  
Leandro Royer ◽  
Bradley S. Launikonis ◽  
Adom González ◽  
...  
Keyword(s):  
Ryanodine Receptors ◽  
Critical Role ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Dihydropyridine Receptors ◽  
Skeletal Muscle Contraction ◽  
Probable Role ◽  
Emission Ratios

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice ( mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 μM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks.

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