Rationale Activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. Objective To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K+ channel underlying mitoKATP. Methods and Results Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and confirmed that ROMK mRNA is present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal and a full length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoKATP activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, shRNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide activated, component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, while knockdown of the native ROMK exacerbates cell death. Conclusions The findings support ROMK as the pore-forming subunit of the cytoprotective mitoKATP channel.

H874 -H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O 2 ·Ϫ ), hydrogen peroxide (H2O2), and hydroxyl radical (HO˙), each of which has been proposed as the signaling ROS. Feedback activation of mitoK ATP provides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO˙excludes H2O2 as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO˙and that it is probably a product of phospholipid oxidation. mitochondria; cardioprotection; reactive oxygen species; KATP channels; cardiac ischemia; ROS signaling REACTIVE OXYGEN SPECIES (ROS) are second messengers of preconditioning (21) and have long been known to be required for cardioprotective signaling (3,10,18,34,43,49). The mechanism of increased ROS is reasonably well understood: signaling from the plasma membrane leads to opening of the mitochondrial ATP-sensitive K ϩ channel (mitoK ATP ; Ref. 19), and the increased K ϩ influx into the matrix causes an increase in ROS, which derive during normoxia from complex I of the respiratory chain (1).The ROS transformations that take place in mitochondria are summarized in Figs. 1 (27). The consequences of ROS signaling for mitochondrial physiology are that two mitochondrial protein kinase-Cε (PKCε) are activated by oxidation of their thiol groups (28). Activation of PKCε1, which is associated with mitoK ATP at the mitochondrial inner membrane, leads to opening of mitoK ATP (13,25). Activation of PKCε2 leads to inhibition of the mitochondrial permeability transition (MPT; Ref. 12). Progress is being made in these areas and in the molecular identification of mitoK ATP (17). However, the ROS responsible for activating these PKCεs in vitro or in vivo is still not known. Because the signaling ROS not only originates in mitochondria but also acts on mitochondria, it should be possible to narrow the search for the signaling ROS by studies on isolated rat heart mitochondria. The objective of these studies was to identify the point in the reaction sequence of ROS transformations at which the ROS signal is formed and, ultimately, to determine the identity of the ROS signal itself.We previously described feedback activation of mitoK ATP , in which mitoK ATP opening by a K ATP channel opener ...

Clinical case reports (CCRs) provide an important means of sharing clinical experiences about atypical disease phenotypes and new therapies. However, published case reports contain largely unstructured and heterogeneous clinical data, posing a challenge to mining relevant information. Current indexing approaches generally concern document-level features and have not been specifically designed for CCRs. To address this disparity, we developed a standardized metadata template and identified text corresponding to medical concepts within 3,100 curated CCRs spanning 15 disease groups and more than 750 reports of rare diseases. We also prepared a subset of metadata on reports on selected mitochondrial diseases and assigned ICD-10 diagnostic codes to each. The resulting resource, Metadata Acquired from Clinical Case Reports (MACCRs), contains text associated with high-level clinical concepts, including demographics, disease presentation, treatments, and outcomes for each report. Our template and MACCR set render CCRs more findable, accessible, interoperable, and reusable (FAIR) while serving as valuable resources for key user groups, including researchers, physician investigators, clinicians, data scientists, and those shaping government policies for clinical trials.

Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidative post-translational modifications (O-PTM) of proteomes in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform that encompasses a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine O-PTM in an ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 1655 proteins containing 3324 oxidized cysteine sites by at least one of the following three modifications: reversible cysteine O-PTM, cysteine sulfinylation (CysSOH), and cysteine sulfonylation (CysSOH). Analyzing the hypertrophy signatures that are reproducibly discovered from this computational workflow unveiled four biological processes with increased cysteine O-PTM. Among them, protein phosphorylation, creatine metabolism, and response to elevated Ca pathways exhibited an elevation of cysteine O-PTM in early stages, whereas glucose metabolism enzymes were increasingly modified in later stages, illustrating a temporal regulatory map in cardiac hypertrophy. Our cysteine O-PTM platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, through the extracted molecular signatures, and provides critical mechanistic insights in cardiac hypertrophy. Data are available via ProteomeXchange with identifier PXD010336.