No abstract

Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

Brown dwarfs and giant gas extrasolar planets have cold atmospheres with a rich chemical compositions from which mineral cloud particles form. Their properties, like particle sizes and material composition, vary with height, and the mineral cloud particles are charged due to triboelectric processes in such dynamic atmospheres. The dynamics of the atmospheric gas is driven by the irradiating host star and/or by the rotation of the objects that changes during its lifetime. Thermal gas ionisation in these ultra-cool but dense atmospheres allows electrostatic interactions and magnetic coupling of a substantial atmosphere volume. Combined with a strong magnetic field B Earth , a chromosphere and aurorae might form as suggested by radio and X-ray observations of brown dwarfs. Non-equilibrium processes like cosmic ray ionisation and discharge processes in clouds will increase the local pool of free electrons in the gas. Cosmic rays and lighting discharges also alter the composition of the local atmospheric gas such that tracer molecules might be identified. Cosmic rays affect the atmosphere through air showers in a certain volume which was modelled with a 3D Monte Carlo radiative transfer code to be able to visualise their spacial extent. Given a certain degree of thermal ionisation of the atmospheric gas, we suggest that electron attachment to charge mineral cloud particles is too inefficient to cause an electrostatic disruption of the cloud particles. Cloud particles will therefore not be destroyed by Coulomb explosion for the local temperature in the collisional dominated brown dwarf and giant gas planet atmospheres. However, the cloud particles are destroyed electrostatically in regions with strong gas ionisation. The potential size of such cloud holes would, however, be too small and might occur too far inside the cloud to mimic the effect of, e.g., magnetic field induced star spots.

Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP1) activity has been observed in heart failure (HF), but the regulatory role of this enzyme on intracellular Ca 2þ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP1 activity, we investigated the effect of the PP1 catalytic subunit on Ca 2þ sparks in permeabilized cardiomyocytes. We used wild-type (WT) and transgenic mice in which the highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation. In WT myocytes, where cytosolic Ca 2þ was clamped at 60nM, 2U/ml of PP1 initially increased Ca 2þ spark frequency (CaSpF) by 2.2-fold, followed by a second phase during which CaSpF returned to control. Spark mass was decreased, but due to the high CaSpF, spark-mediated leak was increased by PP1. This was accompanied by depletion of the sarcoplasmic reticulum (SR) Ca 2þ stores, as determined by application of caffeine. Changes in Ca 2þ release and SR Ca 2þ load were prevented by 5uM of okadaic acid, an inhibitor of PP1. S2808A mutant myocytes showed lower resting CaSpF compared to WT (1.1850.23 vs 2.350.35 sparks/100um/s) and 2U/ml of PP1 failed to generate changes in CaSpF as well as in SR Ca 2þ load. A higher concentration of PP1 (10U/ml) increased CaSpF 4-fold compared to control in WT, and 2.8-fold in S2808A cells, indicating a concentration-dependence. Our results suggest that increased intracellular PP1 activity stimulates RyR2-mediated SR Ca 2þ release and that de-phosphorylation of RyR2-S2808 and at least one not yet identified phosphorylation site may be important in RyR2 modulation.

Mitochondrial Ca 2þ regulates a wide variety of physiological processes, including ATP production and cell death. Rapid Ca 2þ uptake into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) complex, which is composed of the pore forming MCU protein, and the regulatory proteins EMRE, and MICU1/MICU2. Currently, the submitochondrial localization and transmembrane orientation of these proteins are either unknown or under debate. We attack these issues using a classical thiol-modification approach. We removed native MCU complex proteins in HEK293 cells using CRISPR/Cas9, and reintroduced mutants containing a single cysteine at defined positions. Treatments of the mitoplast with a bulky, thiol-reactive compound polyethylene glycol (PEG) maleimide would alter the molecular weight of the protein if the engineered Cys is exposed to the intermembrane space. Using this strategy along with other biochemical methods, we demonstrated that (1) MCU adopts an orientation with the signature DIME motif facing intermembrane space (IMS), ( 2) the conserved C-terminal polyaspartic tail of EMRE is in the IMS, (3) both MICU1 and MICU2 are associated with the outer leaflet of the inner membrane, and (4) although MICU1 is a peripheral membrane protein, MICU2 is an integral component of the inner membrane. Then, applying domain interaction analysis and mutagenesis screening, we identified molecular contacts that govern the Ca 2þ transport behavior of the MCU complex. In particular, we demonstrate that EMRE interacts with MCU through the transmembrane helices to activate the Ca 2þ pore, while using the polyaspartic tail to recruit MICU1/2 to gate the pore. This dual functionality of EMRE ensures that all functional MCU complexes respond appropriately to Ca 2þ stimuli from the cytosol, safeguarding against dangerous Ca 2þ leakage, which could diminish mitochondrial energy output and potentially trigger apoptotic cell death.