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.

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.

scholarly journals Muscarinic receptors stimulate AC2 by novel phosphorylation sites, whereas Gβγ subunits exert opposing effects depending on the G-protein source

2012 ◽  
Vol 447 (3) ◽  
pp. 393-405 ◽  
Author(s):  
Jia X. Shen ◽  
Sebastian Wachten ◽  
Michelle L. Halls ◽  
Katy L. Everett ◽  
Dermot M. F. Cooper
Keyword(s):  
Muscarinic Receptor ◽  
Somatostatin Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Canonical Pathways ◽  
Pkc Protein Kinase C ◽  
Opposite Manner ◽  
Opposing Effects

Direct phosphorylation of AC2 (adenylyl cyclase 2) by PKC (protein kinase C) affords an opportunity for AC2 to integrate signals from non-canonical pathways to produce the second messenger, cyclic AMP. The present study shows that stimulation of AC2 by pharmacological activation of PKC or muscarinic receptor activation is primarily the result of phosphorylation of Ser490 and Ser543, as opposed to the previously proposed Thr1057. A double phosphorylation-deficient mutant (S490/543A) of AC2 was insensitive to PMA (phorbol myristic acid) and CCh (carbachol) stimulation, whereas a double phosphomimetic mutant (S490/543D) mimicked the activity of PKC-activated AC2. Putative Gβγ-interacting sites are in the immediate environment of these PKC phosphorylation sites (Ser490 and Ser543) that are located within the C1b domain of AC2, suggesting a significant regulatory importance of this domain. Consequently, we examined the effect of both Gq-coupled muscarinic and Gi-coupled somatostatin receptors. Employing pharmacological and FRET (fluorescence resonance energy transfer)-based real-time single cell imaging approaches, we found that Gβγ released from the Gq-coupled muscarinic receptor or Gi-coupled somatostatin receptors exert inhibitory or stimulatory effects respectively. These results underline the sophisticated regulatory capacities of AC2, in not only being subject to regulation by PKC, but also and in an opposite manner to Gβγ subunits, depending on their source.

scholarly journals Cardiac Myocyte Cell Cycle Control in Development, Disease, and Regeneration

2007 ◽  
Vol 87 (2) ◽  
pp. 521-544 ◽  
Author(s):  
Preeti Ahuja ◽  
Patima Sdek ◽  
W. Robb MacLellan
Keyword(s):  
Cell Cycle ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Cell Cycle Control ◽  
Cardiac Regeneration ◽  
Perinatal Period ◽  
Fetal Life ◽  
Cell Cycle Reentry ◽  
Adult Cardiac Myocytes ◽  
Species Specific

Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle soon after birth in mammals. Although the extent to which adult cardiac myocytes are capable of cell cycle reentry is controversial and species-specific differences may exist, it appears that for the vast majority of adult cardiac myocytes the predominant form of growth postnatally is an increase in cell size (hypertrophy) not number. Unfortunately, this limits the ability of the heart to restore function after any significant injury. Interest in novel regenerative therapies has led to the accumulation of much information on the mechanisms that regulate the rapid proliferation of cardiac myocytes in utero, their cell cycle exit in the perinatal period, and the permanent arrest (terminal differentiation) in adult myocytes. The recent identification of cardiac progenitor cells capable of giving rise to cardiac myocyte-like cells has challenged the dogma that the heart is a terminally differentiated organ and opened new prospects for cardiac regeneration. In this review, we summarize the current understanding of cardiomyocyte cell cycle control in normal development and disease. In addition, we also discuss the potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration.

Cell-cell contact between adult rat cardiac myocytes regulates Kv1.5 and Kv4.2 K+channel mRNA expression

1998 ◽  
Vol 275 (6) ◽  
pp. C1473-C1480 ◽  
Author(s):  
Kenneth M. Hershman ◽  
Edwin S. Levitan
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Cardiac Myocytes ◽  
Cell Density ◽  
Cardiac Myocyte ◽  
Live Cells ◽  
K Channel ◽  
Cell Contact ◽  
Adult Cardiac Myocytes ◽  
Cell Cell

Regulation of voltage-gated K+channel genes represents an important mechanism for modulating cardiac excitability. Here we demonstrate that expression of two K+channel mRNAs is reciprocally controlled by cell-cell interactions between adult cardiac myocytes. It is shown that culturing acutely dissociated rat ventricular myocytes for 3 h results in a dramatic downregulation of Kv1.5 mRNA and a modest upregulation of Kv4.2 mRNA. These effects are specific, because similar changes are not detected with other channel mRNAs. Increasing myocyte density promotes maintenance of Kv1.5 gene expression, whereas Kv4.2 mRNA expression was found to be inversely proportional to cell density. Conditioned culture medium did not mimic the effects of high cell density. However, paraformaldehyde-fixed myocytes were comparable to live cells in their ability to influence K+channel message levels. Thus the reciprocal effects of cell density on the expression of Kv1.5 and Kv4.2 genes are mediated by direct contact between adult cardiac myocytes. These findings reveal for the first time that cardiac myocyte gene expression is influenced by signaling induced by cell-cell contact.

Abstract P193: Nuclear Localization of the α1B-Adrenergic Receptor Subtype Is Required for Hypertrophic Signaling in Cardiac Myocytes

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Steven C Wu ◽  
Andrew L Cypher ◽  
Chastity L Healy ◽  
Casey D Wright ◽  
Yuan Huang ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Nuclear Localization ◽  
Adrenergic Receptors ◽  
Cardiac Myocyte ◽  
Nuclear Localization Sequence ◽  
Gene Marker ◽  
Receptor Subtype ◽  
Wild Type ◽  
Adult Cardiac Myocytes ◽  
Hypertrophic Signaling

We previously demonstrated that α1-adrenergic receptors (α1-ARs) in the heart are required for physiologic hypertrophy during development and prevent a maladaptive response to pathologic stress. We have also shown that the major subtypes, α1A and α1B, both localize to the nucleus in adult cardiac myocytes. Importantly, we have defined a nuclear α1A-ERK survival and an α1A-PKCδ-cTnI inotropic signaling pathway that both originate at the nucleus and are transduced to cytosolic targets, suggesting that the α1A is required for cardiac myocyte survival and contractility. However, less is known about the molecular function of the α1B. In the current study, we examined the role of the α1B-subtype in hypertrophic signaling. First, we identified a bi-partite nuclear localization sequence (NLS) in the carboxy-terminal tail of the receptor. Mutation of the NLS (α1B-NLSmut) disrupted its localization to the nucleus when expressed in adult cardiac myocytes. We then compared hypertrophic signaling of the wild-type α1B- to the mutated receptor by reconstitution in cardiac myocytes lacking endogenous α1B (α1BKO) receptors. Activation of the wild-type receptor by the α1-agonist phenylephrine in α1BKO myocytes restored hypertrophic signaling, as we observed increased phosphorylation of protein kinase C (PKC) isoforms δ and ε, and histone deacetylases (HDAC) 4 and 5. We also observed increased expression of the hypertrophic gene marker, atrial natriuretic factor (ANF). Expression of the α1B-NLSmut failed to activate hypertrophic signaling in α1BKO cardiac myocytes despite phenylephrine stimulation. Together, our data show that nuclear localization of the α1B-subtype is required for hypertrophic signaling and overall further suggest that α1-adrenergic receptors are functional only at the nucleus in cardiac myocytes.

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