Mitochondrial ion channels/transporters as sensors and regulators of cellular redox signaling.

SIGNIFICANCE: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. RECENT ADVANCES: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. CRITICAL ISSUES: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. FUTURE DIRECTIONS: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters

Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (\u3c30 \u3es) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (\u3c30 \u3es), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (\u3c30 \u3es) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system

Molecular and functional identification of a mitochondrial ryanodine receptor in neurons.

Mitochondrial Ca(2+) controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca(2+) signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca(2+) accumulation is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU), but recent reports also indicate that mitochondrial Ca(2+)-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca(2+)-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca(2+) uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca(2+)-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca(2+) through this channel in response to cytosolic Ca(2+) elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca(2+) uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca(2+) uptake mechanism in response to the elevation of [Ca(2+)]c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca(2+)-mediated functions such as energy metabolism

Pharmacological Modulation of Mitochondrial Ca2+ Content Regulates Sarcoplasmic Reticulum Ca2+ Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species

In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca2+ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca2+ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca2+-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca2+ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca2+ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na+/Ca2+ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca2+ accumulation monitored by mitochondrial-targeted Ca2+ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca2+ imaging showed that accelerated Ca2+ accumulation reduced Ca2+ transient amplitude and promoted generation of spontaneous Ca2+ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca2+ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca2+ imaging demonstrated that enhancement of mitochondrial Ca2+ accumulation further perturbed global Ca2+ handling, increasing the number of cells exhibiting spontaneous Ca2+ waves, shortening RyR refractoriness and decreasing SR Ca2+ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca2+ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca2+ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca2+ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca2+ accumulation can be protective

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council