scholarly journals Ophiopogonin D Increases SERCA2a Interaction with Phospholamban by Promoting CYP2J3 Upregulation

2020 ◽  
Vol 2020 ◽  
pp. 1-22
Author(s):  
Jia Wang ◽  
Wenting You ◽  
Ningning Wang ◽  
Wei Zhou ◽  
Yunxuan Ge ◽  
...  
Keyword(s):  
Heart Failure ◽  
Protective Action ◽  
Chinese Herb ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Epoxyeicosatrienoic Acids ◽  
Specific Mechanism ◽  
Rat Cardiomyocytes ◽  
Beneficial Effects ◽  
Intracellular Target

Ophiopogonin D (OPD), a compound from the Chinese herb Radix Ophiopogonis, reportedly induces increased levels of cytochrome P450 2J3 (CYP2J3)/epoxyeicosatrienoic acids (EETs) and Ca2+ in rat cardiomyocytes. Little is known regarding the specific mechanism between CYP2J3 and Ca2+ homeostasis. Here, we investigated whether CYP2J3 is involved in the protective action of OPD on the myocardium by activating the Ca2+ homeostasis-related protein complex (SERCA2a and PLB) in H9c2 rat cardiomyoblast cells. The interaction between SERCA2a and PLB was measured using fluorescence resonance energy transfer. OPD attenuated heart failure and catalyzed the active transport of Ca2+ into the sarcoplasmic reticulum by inducing the phosphorylation of PLB and promoting the SERCA2a activity. These beneficial effects of OPD on heart failure were abolished after knockdown of CYP2J3 in a model of heart failure. Together, our results identify CYP2J3 as a critical intracellular target for OPD and unravel a mechanism of CYP2J3-dependent regulation of intracellular Ca2+.

scholarly journals β3-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sophie Schobesberger ◽  
Peter T Wright ◽  
Claire Poulet ◽  
Jose L Sanchez Alonso Mardones ◽  
Catherine Mansfield ◽  
...  
Keyword(s):  
Heart Failure ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Soluble Guanylyl Cyclase ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Membrane Structures ◽  
Ion Conductance ◽  
Rat Cardiomyocytes ◽  
Scanning Ion Conductance Microscopy

Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

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.

scholarly journals Thermally Denatured BSA, a Surrogate Additive to Replace BSA in Buffers for High-Throughput Screening

2010 ◽  
Vol 15 (10) ◽  
pp. 1281-1286 ◽  
Author(s):  
Imanol Peña ◽  
Juan Manuel Domínguez
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Negative Impact ◽  
Specific Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Masking Effect ◽  
Binding Properties ◽  
Time Resolved ◽  
Beneficial Effects

The use of thermally denatured bovine serum albumin (tdBSA) as an additive in high-throughput screening (HTS) buffers has been studied with the aim of finding a surrogate to native albumin devoid of its inconveniences, in particular its compound masking effect. The presence of aggregates in the thermally denatured material did not have any negative impact on common readout technologies used in HTS such as fluorescence intensity (FLINT), fluorescence polarization, time-resolved fluorescence resonance energy transfer (TR-FRET) and luminescence. tdBSA rendered the same beneficial effects as native albumin in several assays or even improved its performance due to the lack of specific binding properties. Although tdBSA still binds compounds nonspecifically as any other protein does, it mitigates the compound masking effect observed with native albumin and can be postulated as a convenient surrogate to BSA for HTS purposes.

scholarly journals Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules That Enhance Ca2+ Transport

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1170 ◽  
Author(s):  
Tory M. Schaaf ◽  
Evan Kleinboehl ◽  
Samantha L. Yuen ◽  
Lauren N. Roelike ◽  
Bengt Svensson ◽  
...  
Keyword(s):  
Heart Failure ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Speed ◽  
Large Scale ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Inhibitory Interaction ◽  
Single Polypeptide Chain

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

Abstract 282: Phospholamban Truncation Mutations Including Heart Failure Mutant L39stop Disrupt Membrane Localization.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Neha Abrol ◽  
Nikolai Smolin ◽  
Chris Stefonowicz ◽  
Seth L Robia
Keyword(s):  
Heart Failure ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Full Length ◽  
Membrane Localization ◽  
Minimum Length ◽  
Human Heart Failure ◽  
C Terminus ◽  
Dynamics Simulations ◽  
And Function

Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca 2+ handling and contractility by reversibly inhibiting SR Ca 2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization. Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation. Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization. Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.

P5992Why aortic valve disease may persist after surgery? A joint basic science - Clinical effort

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Petersen ◽  
B Kloth ◽  
N Grammatika-Pavlidou ◽  
T Eschenhagen ◽  
H Reichenspurner ◽  
...  
Keyword(s):  
Heart Failure ◽  
Energy Transfer ◽  
Aortic Valve ◽  
Aortic Valve Disease ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Valve Surgery ◽  
Valve Disease ◽  
Myocardial Tissue ◽  
Aortic Valve Surgery

Abstract Background Diseases of the aortic valve are a common reason for heart surgery. Aortic stenosis (AS) is associated with pressure and aortic regurgitation (AR) with a volume overload of the left ventricle (LV). Over time both pathologies lead to systolic and diastolic heart failure, while progressive downregulation of β-adrenoceptors occurs. While LV re-remodeling occurs in the majority of patients after aortic valve surgery, LV dysfunction persists in one fourth of such patients and leads to a terminal heart failure. We aimed to investigate whether differential remodeling in the protein kinase A (PKA) dependent inotropic response in myocytes and myocardial tissue obtained from patients undergoing aortic valve surgery is associated with the LV re-remodeling after surgery. Methods Preoperatively, pro BNP levels were measured and left ventricular strain analysis via echocardiography was performed. Interventricular septal biopsy was obtained intraoperatively in 10 patients who underwent aortic valve surgery. In-vitro contractility was analyzed in myocardial tissue paced with 4 Hz at 37 °C. Freshly isolated cells were transduced with an adenovirus expressing a cytosolic Förster resonance energy transfer (FRET) based cAMP biosensor (Epac1-camps). After 48 hours of culture, Föster-resonance energy transfer (FRET) was used for the first time to measure cAMP in 60 isolated human ventricular myocytes. Isoprenaline (10 nM – 10 μM) was used for β-adrenoceptor activation and forskolin (10 μmol) to activate adenylyl cyclase directly. Results We found a significantly downregulated β-adrenergic sensitivity in cardiomyocytes of patients with aortic valve disease, although contractile response to forskolin was maintained. Furthermore, we found a clear association between reduced sensitivity to isoprenaline (i.e., high EC50 values) and low maximum effect size to isoprenaline in myocardial tissue of patients with aortic valve disease, pointing out relevant β-adrenoceptor dysfunction. There were no significant differences in basal myocardial force between tissue samples of patients with AR and AS. Conclusion Collectively, our data show a profound remodelling in the cAMP/PKA pathway in patients with aortic valve disease. These disturbances may have an impact on the postoperative ventricular function and possibly on the long-term LV re-remodelling after aortic valve surgery.

scholarly journals Vasodilatory Responses of Renal Interlobular Arteries to Epoxyeicosatrienoic Acids Analog Are Not Enhanced in Ren-2 Transgenic Hypertensive Rats: Evidence Against a Role of Direct Vascular Effects of Epoxyeicosatrienoic Acids in Progression of Experimental Heart Failure

2017 ◽  
pp. 29-39 ◽  
Author(s):  
A. SPORKOVÁ ◽  
Z. HUSKOVÁ ◽  
P. ŠKAROUPKOVÁ ◽  
N. RAMI REDDY ◽  
J. R. FALCK ◽  
...  
Keyword(s):  
Heart Failure ◽  
Renal Function ◽  
Renal Dysfunction ◽  
Epoxyeicosatrienoic Acids ◽  
Sprague Dawley ◽  
Resistance Arteries ◽  
Vascular Effects ◽  
Beneficial Effects ◽  
Function Impairment

Pathophysiological mechanisms underlying the development of renal dysfunction and progression of congestive heart failure (CHF) remain poorly understood. Recent studies have revealed striking differences in the role of epoxyeicosatrienoic acids (EETs), active products of cytochrome P-450-dependent epoxygenase pathway of arachidonic acid, in the progression of aorto-caval fistula (ACF)-induced CHF between hypertensive Ren-2 renin transgenic rats (TGR) and transgene-negative normotensive Hannover Sprague-Dawley (HanSD) controls. Both ACF TGR and ACF HanSD strains exhibited marked intrarenal EETs deficiency and impairment of renal function, and in both strains chronic pharmacologic inhibition of soluble epoxide hydrolase (sEH) (which normally degrades EETs) normalized EETs levels. However, the treatment improved the survival rate and attenuated renal function impairment in ACF TGR only. Here we aimed to establish if the reported improved renal function and attenuation of progression of CHF in ACF TGR observed after sEH blockade depends on increased vasodilatory responsiveness of renal resistance arteries to EETs. Therefore, we examined the responses of interlobar arteries from kidneys of ACF TGR and ACF HanSD rats to EET-A, a new stable 14,15-EET analog. We found that the arteries from ACF HanSD kidneys rats exhibited greater vasodilator responses when compared to the ACF TGR arteries. Hence, reduced renal vasodilatory responsiveness cannot be responsible for the lack of beneficial effects of chronic sEH inhibition on the development of renal dysfunction and progression of CHF in ACF HanSD rats.

scholarly journals Flavonoids enhance rod opsin stability, folding, and self-association by directly binding to ligand-free opsin and modulating its conformation

2019 ◽  
Vol 294 (20) ◽  
pp. 8101-8122 ◽  
Author(s):  
Joseph T. Ortega ◽  
Tanu Parmar ◽  
Beata Jastrzebska
Keyword(s):  
Fruits And Vegetables ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nerve Impulse ◽  
Underlying Mechanism ◽  
Binding Pocket ◽  
Light Signal ◽  
Beneficial Effects ◽  
Ligand Free ◽  
Self Association

Rhodopsin (Rho) is a visual G protein–coupled receptor expressed in the rod photoreceptors of the eye, where it mediates transmission of a light signal into a cell and converts this signal into a nerve impulse. More than 100 mutations in Rho are linked to various ocular impairments, including retinitis pigmentosa (RP). Accordingly, much effort has been directed toward developing ligands that target Rho and improve its folding and stability. Natural compounds may provide another viable approach to such drug discovery efforts. The dietary polyphenol compounds, ubiquitously present in fruits and vegetables, have beneficial effects in several eye diseases. However, the underlying mechanism of their activity is not fully understood. In this study, we used a combination of computational methods, biochemical and biophysical approaches, including bioluminescence resonance energy transfer, and mammalian cell expression systems to clarify the effects of four common bioactive flavonoids (quercetin, myricetin, and their mono-glycosylated forms quercetin-3-rhamnoside and myricetrin) on rod opsin stability, function, and membrane organization. We observed that by directly interacting with ligand-free opsin, flavonoids modulate its conformation, thereby causing faster entry of the retinal chromophore into its binding pocket. Moreover, flavonoids significantly increased opsin stability, most likely by introducing structural rigidity and promoting receptor self-association within the biological membranes. Of note, the binding of flavonoids to an RP-linked P23H opsin variant partially restored its normal cellular trafficking. Together, our results suggest that flavonoids could be utilized as lead compounds in the development of effective nonretinoid therapeutics for managing RP-related retinopathies.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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