Abstract 194: Phosphodiesterase 3 Controls Basal cGMP Levels And Regulates cGMP/cAMP Cross-talk In Adult Mouse Ventricular Cardiomyocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Konrad Götz ◽  
Viacheslav Nikolaev
Keyword(s):  
Cardiac Myocytes ◽  
Cross Talk ◽  
Adult Mouse ◽  
Temporal Dynamics ◽  
Cardiac Contractility ◽  
Resonance Energy ◽  
Direct Stimulation ◽  
Specific Expression ◽  
Cgmp Production ◽  
Adult Cardiac Myocytes

PURPOSE: cGMP is an important second messenger which is involved in the regulation of cardiac contractility and pathological hypertrophy. In cardiomyocytes, signaling by cGMP is organized in microdomains and is considered cardioprotective. Especially in adult cardiac myocytes, measurements of cGMP have been challenging and little is known about the spatio-temporal dynamics of cGMP. Here we developed a transgenic mouse model to visualize cGMP dynamis in adult cardiac myocytes. Methods: We generated transgenic mice with cardiomyocyte-specific expression of a highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5 and performed FRET measurements in freshly isolated adult mouse ventricular myocytes. To analyze cGMP/cAMP crosstalk, FRET experiments were performed in cardiomyocytes isolated from mice transgenically expressing the cAMP sensor Epac1-camps. Results: Basal cytosolic cGMP levels were very low (~10 nM), but could be markedly increased by stimulation with natriuretic peptides (CNP>>ANP). In contrast, direct stimulation of the soluble guanylyl cyclase (sGC) with NO-donors such as SNAP showed no effect. However, constitutive activity of this cyclase contributes to basal cGMP production, since a clear decrease of basal cGMP levels was observed after stimulation with the sGC inhibitor ODQ. This basal cGMP production is regulated by phosphodiesterase (PDE) activity. Unexpectedly, PDE3 is most important in controlling basal cGMP levels, whereas PDE2 and PDE5 are much less active. We could also show that cGMP pools produced by GC-B after CNP stimulation are mainly regulated by PDE3, so that the receptor and this PDE form one functional unit important for the regulation of cGMP/cAMP cross-talk. Conclusion: In summary, we performed the first FRET-based measurements of cGMP in adult cardiomyocytes and we could highlight the key role of PDE3 in the regulation of basal cGMP levels and cGMP/cAMP cross-talk.

Abstract 2375: Regulation of Cardiac Myocyte Contractility by Phospholemman

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jianliang Song ◽  
Xue-Qian Zhang ◽  
JuFang Wang ◽  
Ellina Cheskis ◽  
Tung O Chan ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Adult Mouse ◽  
Surface Membrane ◽  
Cardiac Contractility ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Normal Surface ◽  
Membrane Area ◽  
Myocyte Contractility

Phospholemman (PLM) regulates cardiac contractility by modulating Na + /Ca 2+ exchanger (NCX1) and/or Na + -K + -ATPase activities. PLM, when phosphorylated at serine68, disinhibits Na + -K + -ATPase but inhibits NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 hours had normal surface membrane area and t-tubule appearance, and exhibited near normal contractility. Contractile differences between wild-type (WT) and PLM knockout (KO) myocytes were preserved after 48h of culture. Infection with adenovirus overexpressing GFP did not affect contractility at 48h. When WT PLM was overexpressed in PLM-KO myocytes, part of PLM was phosphorylated, both Na + -K + -ATPase current (I pump ) and Na + /Ca 2+ exchange current (I NaCa ) were depressed, and contractility and [Ca 2+ ] i transients reverted back to those observed in cultured WT myocytes. Overexpressing PLMS68E mutant (phosphomimetic) in PLM-KO myocytes resulted in suppression of I NaCa but no effect on I pump . Contractility and [Ca 2+ ] i transient amplitudes in PLM-KO myocytes overexpressing PLMS68E mutant were depressed when compared to PLM-KO myocytes overexpressing GFP. Overexpressing PLMS68A mutant (mimicking unphosphorylated PLM) in PLM-KO myocytes had no effects on I NaCa but decreased I pump . Contractility and [Ca 2+ ] i transient amplitudes in PLM-KO myocytes overexpressing S68A mutant were similar to PLM-KO myocytes overexpressing GFP. Neither WT PLM nor its mutants had any effect on SR Ca 2+ uptake in KO myocytes. We conclude that at the single myocyte level, PLM affects cardiac contractility and [Ca 2+ ] i homeostasis primarily by its direct inhibitory effects on Na + /Ca 2+ exchange.

β-Adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor

2005 ◽  
Vol 289 (2) ◽  
pp. C455-C461 ◽  
Author(s):  
Sunita Warrier ◽  
Andriy E. Belevych ◽  
Monica Ruse ◽  
Richard L. Eckert ◽  
Manuela Zaccolo ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Cardiac Myocytes ◽  
Adrenergic Receptor ◽  
Cardiac Myocyte ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Functional Responses ◽  
Adult Cardiac Myocytes ◽  
Camp Activity

β-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by β-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of β-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types.

Tumor Necrosis Factor-α Provokes a Hypertrophic Growth Response in Adult Cardiac Myocytes

Circulation ◽  
1997 ◽  
Vol 95 (5) ◽  
pp. 1247-1252 ◽  
Author(s):  
Tomoyuki Yokoyama ◽  
Masayuki Nakano ◽  
John L. Bednarczyk ◽  
Bradley W. McIntyre ◽  
Mark Entman ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Cardiac Myocytes ◽  
Tumor Necrosis ◽  
Growth Response ◽  
Tumor Necrosis Factor Α ◽  
Hypertrophic Growth ◽  
Adult Cardiac Myocytes ◽  
Factor Α ◽  
Necrosis Factor

scholarly journals Tissue-Restricted Expression of the Cardiac α-Myosin Heavy Chain Gene Is Controlled by a Downstream Repressor Element Containing a Palindrome of Two Ets-Binding Sites

1998 ◽  
Vol 18 (12) ◽  
pp. 7243-7258 ◽  
Author(s):  
Madhu Gupta ◽  
Radovan Zak ◽  
Towia A. Libermann ◽  
Mahesh P. Gupta
Keyword(s):  
Gene Regulation ◽  
Myosin Heavy Chain ◽  
Cardiac Myocytes ◽  
Heavy Chain ◽  
Binding Sites ◽  
Cardiac Myocyte ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Nonmuscle Cells ◽  
Muscle Gene

ABSTRACT The expression of the α-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in α-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac α-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the α-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the α-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the α-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the α-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the α-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the α-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the α-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.

scholarly journals Hypoxia Delays the Intracellular Ca2+Clearance by Na+- Ca2+Exchanger in Human Adult Cardiac Myocytes

2001 ◽  
Vol 42 (3) ◽  
pp. 333 ◽  
Author(s):  
Seung Il Park ◽  
Eun Ju Park ◽  
Nak Hyun Kim ◽  
Wan Ki Baek ◽  
Young Tak Lee ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Human Adult ◽  
Adult Cardiac Myocytes

Protein kinase C-ζ modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt

2002 ◽  
Vol 282 (1) ◽  
pp. H320-H327 ◽  
Author(s):  
Yukitaka Shizukuda ◽  
Peter M. Buttrick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Adult Rat ◽  
Kinase C ◽  
Adult Cardiac Myocytes ◽  
Pkc Ζ

We hypothesized that thromboxane A2 (TxA2) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a TxA2 mimetic [1S-[1α,2α(Z),3β(1E,3S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a TxA2 receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. TxA2 stimulation resulted in membrane translocation of PKC-ζ but not PKC-α, -βII, -δ, and -ε at 3 min and 1 h. The activation of PKC-ζ by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-ζ pseudosubstrate peptide (ζ-PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with ζ-PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of ζ-PS. Therefore, our results suggest that the activation of PKC-ζ modulates TxA2 receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes.

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