SUMMARY The mitochondrion is the primary source of reactive oxygen species (ROS) in eukaryotic cells. With the aid of a novel mitochondrial matrix-targeted superoxide indicator, here we show that individual mitochondria undergo spontaneous bursts of superoxide generation, termed “superoxide flashes”. Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and reflect elementary events of superoxide production within single mitochondria across a wide diversity of cells. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore (mPTP) and are fueled by electron transfer complexes-dependent superoxide production. While decreased during cardiac hypoxia/anoxia, a flurry of superoxide flash activity contributes to the destructive rebound ROS burst observed during early reoxygenation after anoxia. The discovery of superoxide flashes reveals a novel mechanism for quantal ROS production by individual mitochondria and substantiates the central role of mPTP in oxidative stress related pathology in addition to its well-known role in apoptosis.

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual‐specificity MAP kinase phosphatases typified by CL100/MKP‐1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos‐1 cells. Like CL100/ MKP‐1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos‐1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress‐activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress‐mediated activation of the JNK‐1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual‐specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.

Skeletal muscle contractions are initiated by an increase in Ca 2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca 2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca 2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca 2+ in internal stores, which is consistent with increased SR luminal Ca 2+ . These findings define critical roles for Stac3 in EC coupling and human disease.zebrafish | skeletal muscle | excitation-contraction coupling | dihydropyridine receptor | Native American myopathy

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

However, Q max was increased 85% 6 days after injection of dyspedic myotubes with cDNA encoding the wild-type RyR-1 but not E4032A. Because normal and dyspedic myotubes exhibited a similar density of T-type Ca 2؉ current (T-current), the presence of RyR-1 does not appear to cause a general overall increase in protein synthesis. Thus, long-term expression of L-channels in skeletal myotubes is promoted by Ca 2؉ released through RyRs occurring either spontaneously or during excitationcontraction coupling.The skeletal muscle dihydropyridine receptor (DHPR) 1 functions both as a slowly activating L-type Ca 2ϩ channel (L-channel) and as a voltage sensor that controls the activity of the type 1 ryanodine receptor (RyR-1) present in the sarcoplasmic reticulum (SR). During excitation-contraction (EC) coupling, sarcolemmal depolarization (e.g. an action potential) induces voltage-driven conformational changes in the DHPR, which can be measured electrophysiologically as nonlinear capacitative currents, termed intramembrane charge movements or gating currents. These charge movements are thought to mechanically activate RyR-1 proteins during EC coupling and thus lead to a massive release of SR calcium (orthograde signal of EC coupling; see Ref. 1 for review).Analysis of skeletal myotubes derived from RyR-1-knockout (dyspedic) mice has revealed that in addition to the orthograde signal of EC coupling (signal transmitted from the DHPR to the RyR-1), there is also a retrograde signal whereby RyR-1 promotes the calcium conducting activity of the skeletal L-channel (2, 3). This conclusion was inferred from the observation that despite a significant surface density of DHPRs, dyspedic myotubes exhibit a marked (ϳ90%) reduction in L-current. Moreover, short-term (2-4 days) expression of RyR-1 in dyspedic myotubes considerably enhances L-current density in the absence of a change in intramembrane charge movement (2, 3). These observations indicate that RyR-1 promotes the L-channel activity of the skeletal muscle DHPR in a manner that is independent of L-channel expression (retrograde signal of EC coupling).Dyspedic muscle exhibits a 25-50% reduction in total DHP binding (4, 5). Accordingly, we have reported that dyspedic myotubes possess a significant reduction in maximal intramembrane charge movement compared with normal myotubes (3). This apparent reduction in the number of functional DHPRs in the sarcolemma cannot completely account for the ϳ90% decrease in L-current density found in dyspedic myotubes. In fact, dyspedic myotubes exhibit a nearly 5-fold reduction in the current-to-charge and conductance-to-charge (G max / Q max ) ratios, compared with both normal and RyR-1-expressing dyspedic myotubes (3). These observations support the conclusion of Nakai et al. (2) that RyR-1 promotes the Ca 2ϩ conducting activity of the skeletal muscle L-channel. However, the mechanism(s) underlying the different DHPR expression levels in normal and dyspedic muscle have yet to be investigated. Considering the functional effects of reintroduction...

Myotubular myopathy (MTM) is a severe X-linked disease without existing therapies. Here, we show that tamoxifen ameliorates MTM-related histopathological and functional abnormalities in mice, and nearly doubles survival. The beneficial effects of tamoxifen are mediated primarily via estrogen receptor signaling, as demonstrated through in vitro studies and in vivo phenotypic rescue with estradiol. RNA sequencing and protein expression analyses revealed that rescue is mediated in part through post-transcriptional reduction of dynamin-2, a known MTM modifier. These findings demonstrate an unexpected ability of tamoxifen to improve the murine MTM phenotype, providing preclinical evidence to support clinical translation.

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BACKGROUND-Mutations in the type I ryanodine receptor gene (RYR1) result in malignant hyperthermia, a pharmacogenetic disorder typically triggered by administration of anesthetics. However, cases of sudden death during exertion, heat challenge, and febrile illness in the absence of triggering drugs have been reported. The underlying causes of such drug-free fatal "awake" episodes are unknown. METHODS-De novo R3983C variant in RYR1 was identified in two unrelated children that experienced fatal, nonanesthetic awake episodes associated with febrile illness and heat stress. One of the children also possessed a second novel maternally-inherited D4505H variant located on a separate haplotype. Effects of all possible heterotypic expression conditions on RYR1 sensitivity to caffeine-induced Ca 2+ release were determined in expressing RyR1-null myotubes. RESULTS-Compared to wild-type RYR1 alone (EC 50 = 2.85 ± 0.49 mM), average (±SEM) caffeine sensitivity of Ca 2+ release was modestly increased following coexpression with either R3983C (EC 50 = 2.00 ± 0.39 mM) or D4505H (EC 50 = 1.64 ± 0.24 mM). Remarkably,