Mitofusin 2 is essential for IP3-mediated SR/Mitochondria metabolic feedback in ventricular myocytes

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP$_3$) by activating their membrane-bound receptors. We have previously demonstrated that IP$_3$-mediated sarcoplasmic reticulum (SR) Ca$^{2+}$ release results in mitochondrial Ca$^{2+}$ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP$_3$-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca$^{2+}$ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca$^{2+}$ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca$^{2+}$ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca$^{2+}$ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca$^{2+}$ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca$^{2+}$ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP$_3$-mediated SR Ca$^{2+}$ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP$_3$R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter)

Mitofusin 2 is essential for IP3-mediated SR/mitochondria metabolic feedback in ventricular myocytes

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP$_3$) by activating their membrane-bound receptors. We have previously demonstrated that IP$_3$-mediated sarcoplasmic reticulum (SR) Ca$^{2+}$ release results in mitochondrial Ca$^{2+}$ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP$_3$-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca$^{2+}$ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca$^{2+}$ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca$^{2+}$ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca$^{2+}$ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca$^{2+}$ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca$^{2+}$ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP$_3$-mediated SR Ca$^{2+}$ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP$_3$R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter)

Quantum Fluctuations and the Unruh Effect in Strongly-Coupled Conformal Field Theories

Through the AdS/CFT correspondence, we study a uniformly accelerated quark in the vacuum of strongly-coupled conformal field theories in various dimensions, and determine the resulting stochastic fluctuations of the quark trajectory. From the perspective of an inertial observer, these are quantum fluctuations induced by the gluonic radiation emitted by the accelerated quark. From the point of view of the quark itself, they originate from the thermal medium predicted by the Unruh effect. We scrutinize the relation between these two descriptions in the gravity side of the correspondence, and show in particular that upon transforming the conformal field theory from Rindler space to the open Einstein universe, the acceleration horizon disappears from the boundary theory but is preserved in the bulk. This transformation allows us to directly connect our calculation of radiation-induced fluctuations in vacuum with the analysis by de Boer et al. of the Brownian motion of a quark that is on average static within a thermal medium. Combining this same bulk transformation with previous results of Emparan, we are also able to compute the stress-energy tensor of the Unruh thermal medium.Comment: 1+31 pages; v2: reference adde

Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated.Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively.Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 μm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4–5 μm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB β-hydroxybutyrate (β-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where β-OHB further increased the number of cells with giant Parkin-rich regions. This β-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria.Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade

Very accurate cryogenic mechanisms for CRIRES+

After 5 years of operation on the VLT, a large upgrade of CRIRES (the ESO Cryogenic InfraRed Echelle Spectrograph) was decided mainly in order to increase the efficiency. Using a cross dispersion design allows better wavelength coverage per exposure. This means a complete re-design of the cryogenic pre-optic which were including a predispersion stage with a large prism as dispersive element. The new design requires a move of the entrance slit and associated decker toward the first intermediate focal plane right behind the window. Implement 2 functions with high positioning accuracy in a pre-defined and limited space was a real challenge. The design and the test results recorded in the ESO Cryogenic Test Facility are reported in this paper. The second critical function is the grating wheel which positions the 6 cross disperser gratings into the beam. The paper describes the design of the mechanism which includes a detente system in order to guaranty the 5 arc sec positioning reproducibility requested. The design includes also feedback system, based on switches, in order to ensure that the right grating is in position before starting a long exposure. The paper reports on the tests carried out at cryogenic temperature at the sub-system level. It also includes early performances recorded in the instrument along the first phases of the system test

A unique infrared spectropolarimetric unit for CRIRES+

High-resolution infrared spectropolarimetry has many science applications in astrophysics. One of them is measuring weak magnetic fields using the Zeeman effect. Infrared domain is particularly advantageous as Zeeman splitting of spectral lines is proportional to the square of the wavelength while the intrinsic width of the line cores increases only linearly. Important science cases include detection and monitoring of global magnetic fields on solar-type stars, study of the magnetic field evolution from stellar formation to the final stages of the stellar life with massive stellar winds, and the dynamo mechanism operation across the boundary between fully- and partially-convective stars. CRIRES+ (the CRIRES upgrade project) includes a novel spectropolarimetric unit (SPU) based on polar- ization gratings. The novel design allows to perform beam-splitting very early in the optical path, directly after the tertiary mirror of the telescope (the ESO Very Large Telescope, VLT), minimizing instrumental polariza- tion. The new SPU performs polarization beam-splitting in the near-infrared while keeping the telescope beam mostly unchanged in the optical domain, making it compatible with the adaptive optics system of the CRIRES+ instrument. The SPU consists of four beam-splitters optimized for measuring circular and linear polarization of spectral lines in YJ and HK bands. The SPU can perform beam switching allowing to correct for throughput in each beam and for variations in detector pixel sensitivity. Other new features of CRIRES+, such as substantially increased wavelength coverage, stability and advanced data reduction pipeline will further enhance the sensitivity of the polarimetric mode. The combination of the SPU, CRIRES+ and the VLT is a unique facility for making major progress in understanding stellar activity. In this article we present the design of the SPU, laboratory measurements of individual components and of the whole unit as well as the performance prediction for the operation at the VLT

Full system test and early preliminary acceptance Europe results for CRIRES+

CRIRES+ is the new high-resolution NIR echelle spectrograph intended to be operated at the platform B of VLT Unit telescope UT3. It will cover from Y to M bands (0.95-5.3um) with a spectral resolution of R = 50000 or R=100000. The main scientific goals are the search of super-Earths in the habitable zone of low-mass stars, the characterisation of transiting planets atmosphere and the study of the origin and evolution of stellar magnetic fields. Based on the heritage of the old adaptive optics (AO) assisted VLT instrument CRIRES, the new spectrograph will present improved optical layout, a new detector system and a new calibration unit providing optimal performances in terms of simultaneous wavelength coverage and radial velocity accuracy (a few m/s). The total observing efficiency will be enhanced by a factor of 10 with respect to CRIRES. An innovative spectro-polarimetry mode will be also offered and a new metrology system will ensure very high system stability and repeatability. Fiinally, the CRIRES+ project will also provide the community with a new data reduction software (DRS) package. CRIRES+ is currently at the initial phase of its Preliminary Acceptance in Europe (PAE) and it will be commissioned early in 2019 at VLT. This work outlines the main results obtained during the initial phase of the full system test at ESO HQ Garching

Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) [e.g. β2-adrenoceptor (β2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β2AR and A2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β2AR- and A2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β2AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A2BR was regulated by GRK5, but not GRK2. β2AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A2BR-AC signalling and attenuated A2BR desensitization, while β2AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle