Role of phosphodiesterases in the development of takotsubo syndrome

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Huebscher ◽  
T Borchert ◽  
G Hasenfuss ◽  
V.O Nikolaev ◽  
K Streckfuss-Boemeke
Keyword(s):  
Resonance Energy ◽  
Induced Pluripotent Stem Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Left Ventricular ◽  
Camp Level ◽  
Camp Signaling ◽  
Patient Specific ◽  
Funding Source ◽  
Takotsubo Syndrome

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. We identified a higher sensitivity to catecholamine-induced stress toxicity as mechanism associated with the TTS phenotype in our former study, but the pathogenesis of TTS is still not completely understood. In this study our aim was to prove the hypothesis of an altered phosphodiesterase (PDE)-dependent 3',5'-cyclic adenosine monophosphate (cAMP)-signaling in TTS in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods and results We generated functional TTS-iPSC-CMs and treated them with catecholamines to mimic a TTS-phenotype. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used Förster resonance energy transfer (FRET) based cAMP sensors, which are specifically located in the cytosol or at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA) micro domain. We demonstrated that β-adrenergic receptor (β-AR) stimulations resulted in stronger cytosolic FRET responses in TTS-CMs compared to controls. In contrast, no differences of cAMP level were observed in the SERCA-PLN micro domain between TTS- and control-iPSC-CMs. To analyze the interplay of β-AR signaling and specific PDE contribution to the cAMP signaling in TTS, specific PDE-inhibitors were used. We were able to show in the cytosol that after β-AR stimulation, the strong effects of the PDE4 family of control cells were significantly decreased in diseased TTS CMs, which is in line with previously described reduced PDE4 activity in failing mouse hearts. In contrast, the contribution of PDE3 to cytoplasmic cAMP degradation was increased in TTS (Figure 1 A). This is in line with increased PDE3A and down-regulated PDE4D protein expression in TTS-iPSC-CMs compared to control cells. Analysis of PDE-dependent cAMP level in the SERCA micro domain show also a significantly reduced PDE4 activity. But the dynamic cytosolic PDE contribution of PDE2 and PDE3 after catecholamine treatment in TTS is lost in SERCA micro domain (Figure1B). Conclusion Our data showed for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. We demonstrated an isozym shift from PDE4 in control to PDE3 and PDE2 in TTS and identified PDE4 as an important player in the β-adrenergic cAMP signaling in TTS. Therefore, PDE4 activators may be a possible new therapeutic target option in the treatment of TTS. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DZHK

3074Analyzing the regulation of a catecholamine-dependent altered cAMP signaling in a patient-specific induced pluripotent stem cell takotsubo-model

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
D Huebscher ◽  
T Borchert ◽  
G Hasenfuss ◽  
V Nikolaev ◽  
K Streckfuss-Boemeke
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Camp Signaling ◽  
Pde4 Inhibitor ◽  
Patient Specific ◽  
Adrenergic Signaling ◽  
Downstream Effects ◽  
Induced Pluripotent

Abstract Background/Purpose Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. Although, we identified an enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced stress toxicity as mechanisms associated with the TTS phenotype in our former study, the pathogenesis of TTS is still not completely understood. Here, we aimed to prove the hypothesis of a phosphodiesterase (PDE)-dependent regulation of 3',5'-cyclic adenosine monophosphate (cAMP) signaling in TTS under catecholamine stress. Methods and results We generated functional TTS induced pluripotent stem cell-derived cardiomyocytes (TTS-iPSC-CMs) from 6 patients and treated the cells with catecholamines to mimic a TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we could observe that β-adrenergic receptor (β-AR) stimulations led to stronger FRET responses in the cytosol of TTS-CMs as compared to controls. Besides β-ARs, PDEs are main players involved in cAMP signaling in CMs. At basal level TTS-CM show a significantly higher PDE3A and a reduced PDE4D protein expression in the TTS-CMs compared to control. In addition, FRET experiments show that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of control cells were significantly decreased in TTS-CMs. This is in line with previously described reduced PDE4 activity in failing mouse hearts. By analyzing PDE-dependent cAMP downstream effects as PKA-dependent phosphorylation, we could show an additional increase of PLN phosphorylation (PLN-S16), especially in control, when treating iPSC-CMs with a combination of iso and PDE4 inhibitor. In contrast, in TTS-iPSC-CMs the contribution of the PDE-families PDE2, 3 or 4 to phosphorylation of PLN-S16 was increased over iso alone. This suggests that different PDEs in TTS and control are involved in functional segregation of the SERCA2a microdomain from the cytosol in terms of cAMP downstream effects. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, we used a SERCA micro domain targeted FRET based cAMP sensor. In contrast to the cytosolic cAMP regulation, the PDE4 inhibitor effects in the SERCA2 micro domain were only slightly decreased in TTS. Instead, the contribution of PDE2 to local cAMP degradation was slightly increased. Conclusion Our data show for the first time alterations of local cAMP signaling in healthy and diseased TTS-iPSC-CMs. TTS leads to changes in PDE composition in the cytosol but not significantly in SERCA microdomain. Our results uncover a PDE-dependent altered β-adrenergic signaling as a potential disease cause. This data highlight that TTS-iPSC-CMs can be used to provide a versatile tool for evaluating new treatment options for TTS as therapeutic targets.

Genetic variants in calcium regulatory cardiac genes and their contribution to Takotsubo syndrome

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
F Treu ◽  
N Dybkova ◽  
P Jung ◽  
Y Li ◽  
D Huebscher ◽  
...  
Keyword(s):  
Genetic Predisposition ◽  
Genetic Variants ◽  
Left Ventricular ◽  
Calcium Currents ◽  
Epifluorescence Microscopy ◽  
Patient Specific ◽  
Funding Source ◽  
Sequencing Analysis ◽  
Takotsubo Syndrome ◽  
Calcium Load

Abstract Background and purpose Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction similar to a myocardial infarction (MI) in the absence of coronary artery stenosis. Patients show symptoms similar to the acute MI with increased biomarkers and blood serum catecholamines. Recently, we developed a patient-specific TTS stem cell model and identified a higher sensitivity to catecholamine-induced stress. Furthermore, familial TTS cases and genetic studies point to a genetic predisposition. The purpose of this study was to analyze a genetic predisposition by characterizing genetic variants in genes associated with cardiac pathologies and their impact on calcium homoeostasis in TTS. Methods and results Whole exome sequencing analysis of a TTS patient discovered 2 missense AHNAK variants in its C-terminal domain and in addition the missense variant F189L in the calcium buffering calsequestrin 2 gene (CASQ2). AHNAK is a 700kDa big nucleoprotein and is involved in the β-adrenergic regulation of the cardiac calcium channel Cav1.2. 3-month old TTS-iPSC-derived cardiomyocytes (CM) were generated and the variants were confirmed by sequencing. We found AHNAK higher expressed in TTS-iPSC-CMs compared to control, whereas no expression alteration was observed for Cav1.2. Since AHNAK is described to act as a repressor towards Cav1.2, which is relieved under β-adrenergic stimulation, we analyzed the effect of AHNAK variants on a potential co-localization and interaction between both proteins. AHNAK and Cav1.2 were shown to co-localize in the cytoplasm as well as the membranes and co-immunoprecipitation experiments confirmed an interaction of AHNAK and Cav1.2 in all tested control- and TTS-iPSC-CMs. On a functional level, we were able to show by patch clamp analysis that Cav1.2 calcium currents are significantly increased in TTS-iPSC-CMs compared to control. The influence of CASQ2-F189L on sarcomeric reticulum (SR) calcium load was analyzed by epifluorescence microscopy using FURA4 and caffeine-applications. We found significantly decreased SR calcium content with an increased fractional release during systole in TTS-iPSC-CMs. To test, whether these variants are the main reason for altered interaction of AHNAK and Cav1.2, calcium currents or SR calcium load in TTS need to be proven in the future by using CRISPR/Cas9-rescued AHNAK/CASQ2 lines. Conclusion Here we show the cardiac functional consequences of AHNAK and CASQ2 missense mutations in TTS-iPSC-CMs with regard to calcium currents and SR calcium load. These results show that AHNAK and CASQ2 variants may predispose to TTS and enable a new therapeutic option for TTS. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Else Kröner-Fresenius Foundation

scholarly journals Cardiac Hypertrophy Changes Compartmentation of cAMP in Non-Raft Membrane Microdomains

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 535
Author(s):  
Nikoleta Pavlaki ◽  
Kirstie A. De Jong ◽  
Birgit Geertz ◽  
Viacheslav O. Nikolaev ◽  
Alexander Froese
Keyword(s):  
Cardiac Hypertrophy ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Pressure Overload ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Regulation ◽  
Membrane Microdomains ◽  
Transgenic Mouse Line

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

Abstract 248: Aberrant TGFβ Signaling as an Etiology of Left Ventricular Non-compaction Cardiomyopathy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Kazuki Kodo ◽  
Sang-Ging Ong ◽  
Fereshteh Jahanbani ◽  
Vittavat Termglinchan ◽  
Kolsoum InanlooRahatloo ◽  
...  
Keyword(s):  
Left Ventricle ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Patient Specific ◽  
Developmental Defect ◽  
Tgfβ Signaling ◽  
Growth Factor Beta ◽  
Induced Pluripotent

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and has a unique phenotype with characteristically extensive hypertrabeculation of the left ventricle, similar to the embryonic left ventricle, suggesting a developmental defect of the embryonic myocardium. However, studying this disease has been challenging due to the lack of an animal model that can faithfully recapitulate the clinical phenotype of LVNC. To address this, we showed that patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from a family with LVNC history recapitulated a developmental defect consistent with the LVNC phenotype at the single-cell level. We then utilized hiPSC-CMs to show that increased transforming growth factor beta (TGFβ) signaling is one of the central mechanisms underlying the pathogenesis of LVNC. LVNC hiPSC-CMs demonstrated decreased proliferative capacity due to abnormal activation of TGFβ signaling (Figs A-B). Exome sequencing demonstrated a mutation in TBX20, which regulates TGFβ signaling through upregulation of ITGAV, contributing to the LVNC phenotype. Inhibition of abnormal TGFβ signaling or genetic correction of the TBX20 mutation (Figs C-D) using TALEN reversed the proliferation defects seen in LVNC hiPSC-CMs. Our results demonstrate that hiPSC-CMs are a useful tool for the exploration of novel mechanisms underlying poorly understood cardiomyopathies such as LVNC. Here we provide the first evidence of activation of TGFβ signaling as playing a role in the pathogenesis of LVNC.

scholarly journals Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Kaley M. Wilburn ◽  
Christine R. Montague ◽  
Bo Qin ◽  
Ashley K. Woods ◽  
Melissa S. Love ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Adenylyl Cyclase ◽  
Cholesterol Metabolism ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Camp Signaling ◽  
Camp Synthesis ◽  
Pharmacokinetic Properties ◽  
Bacterial Utilization

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

GPER mediates estrogen cardioprotection against epinephrine-induced stress

2021 ◽  
Author(s):  
Lu Fu ◽  
Hongyuan Zhang ◽  
Jeremiah Ong’achwa Machuki ◽  
Tingting Zhang ◽  
Lin Han ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Protective Role ◽  
Left Ventricular ◽  
Internal Diameter ◽  
High Dose ◽  
Adult Rats ◽  
Rat Cardiomyocytes ◽  
Lactate Dehydrogenase Release

Currently, there are no conventional treatments for stress-induced cardiomyopathy (SCM, also known as Takotsubo syndrome), and the existing therapies are not effective. The recently discovered G protein- coupled estrogen receptor (GPER) executes the rapid effects of estrogen (E2). In this study, we investigated the effects and mechanism of GPER on epinephrine (Epi)-induced cardiac stress. SCM was developed with a high dose of Epi in adult rats and human-induced pluripotent stem cells–derived cardiomyocytes(hiPSC-CMs). (1) GPER activation with agonist G1/ E2 prevented an increase in left ventricular internal diameter at end-systole, the decrease both in ejection fraction and cardiomyocyte shortening amplitude elicited by Epi. (2) G1/ E2 mitigated heart injury induced by Epi, as revealed by reduced plasma brain natriuretic peptide and lactate dehydrogenase release into culture supernatant. (3) G1/E2 prevented the raised phosphorylation and internalization of β2-adrenergic receptors（β2AR）. (4) Blocking Gαi abolished the cardiomyocyte contractile inhibition by Epi. G1/E2 downregulated Gαi activity of cardiomyocytes and further upregulated cyclic adenosine monophosphate concentration in culture supernatant treated with Epi. (5) G1/E2 rescued decreased Ca2+ amplitude and Ca2+ channel current (ICa-L) in rat cardiomyocytes. Notably, the above effects of E2 were blocked by the GPER antagonist, G15. In hiPSC-CM (which expressed GPER, β1AR and β2ARs), knockdown of GPER by siRNA abolished E2 effects on increasing ICa-L and action potential duration in the stress state. In conclusion, GPER played a protective role against SCM. Mechanistically, this effect was mediated by balancing the coupling of β2AR to the Gαs and Gαi signalling pathways.

scholarly journals Emerging themes of cAMP regulation of the pulmonary endothelial barrier

2011 ◽  
Vol 300 (5) ◽  
pp. L667-L678 ◽  
Author(s):  
Sarah L. Sayner
Keyword(s):  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Signaling Cascades ◽  
Camp Signaling ◽  
Endothelial Barrier ◽  
Adenylyl Cyclases ◽  
Cortical Actin ◽  
Membrane Compartment ◽  
Barrier Regulation ◽  
Actomyosin Contraction

The presence of excess fluid in the interstitium and air spaces of the lung presents severe restrictions to gas exchange. The pulmonary endothelial barrier regulates the flux of fluid and plasma proteins from the vascular space into the underlying tissue. The integrity of this endothelial barrier is dynamically regulated by transitions in cAMP (3′,5′-cyclic adenosine monophosphate), which are synthesized in discrete subcellular compartments. Cyclic AMP generated in the subplasma membrane compartment acts through PKA and Epac (exchange protein directly activated by cAMP) to tighten cell adhesions, strengthen cortical actin, reduce actomyosin contraction, and decrease permeability. Confining cAMP within the subplasma membrane space is critical to its barrier-protective properties. When cAMP escapes the near membrane compartment and gains access to the cytosolic compartment, or when soluble adenylyl cyclases generate cAMP within the cytosolic compartment, this second messenger activates established cytosolic cAMP signaling cascades to perturb the endothelial barrier through PKA-mediated disruption of microtubules. Thus the concept of cAMP compartmentalization in endothelial barrier regulation is gaining momentum and new possibilities are being unveiled for cytosolic cAMP signaling with the emergence of the bicarbonate-regulated mammalian soluble adenylyl cyclase (sAC or AC10).

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