Smooth muscle 22α deficiency impairs oxytocin-induced uterine contractility in mice at full-term pregnancy.

Smooth muscle 22α (SM22α, namely Transgelin), as an actin-binding protein, regulates the contractility of vascular smooth muscle cells (VSMCs) by modulation of the stress fiber formation. However, little is known about the roles of SM22α in the regulation of uterine contraction during parturition. Here, we showed that contraction in response to oxytocin (OT) was significantly decreased in the uterine muscle strips from SM22α knockout (Sm22α-KO) mice, especially at full-term pregnancy, which may be resulted from impaired formation of stress fibers. Furthermore, serious mitochondrial damage such as the mitochondrial swelling, cristae disruption and even disappearance were observed in the myometrium of Sm22α-KO mice at full-term pregnancy, eventually resulting in the collapse of mitochondrial membrane potential and impairment in ATP synthesis. Our data indicate that SM22α is necessary to maintain uterine contractility at delivery in mice, and acts as a novel target for preventive or therapeutic manipulation of uterine atony during parturition.

Identification and characterization of Letm1 gene in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis. Previous studies have shown that the perturbation of mitochondrial metabolism in T. gondii results in growth deficiency in host cells and lack of virulence in animals. Members of this Letm1 protein family are inner mitochondrial membrane proteins which play a role in potassium and hydrogen ion exchange. Letm1 has not been characterized in T. gondii. In this study, a potential TgLetm1 gene (TgGT1_288400) with Letm1-like protein domain coding sequence was identified in T. gondii. Indirect immunofluorescence assays suggested that TgLetm1 localized to the mitochondria in tachyzoites, as indicated by the colocalization with mitochondrial marker Mitotracker. TgLetm1 was found in the membrane fraction by western blot analysis. To investigate the role of TgLetm1 in T. gondii, we generated a tetracycline-inducible TgLetm1-knock-down mutant. The conditional deletion of TgLetm1 resulted in mitochondrial swelling. Functional studies showed that the conditional deletion of TgLetm1 resulted in growth inhibition, deficiency in invasion and replication, and lack of virulence in mice.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Simple kinetic model of mitochondrial swelling in cardiac cells.

Mitochondria play an important role in both cell survival and cell death. In response to oxidative stress, they undergo opening of non-selective permeability transition pores (PTP) in the inner mitochondrial membrane. Sustained PTP opening triggers mitochondrial swelling due to increased colloidal osmotic pressure in the matrix accompanied by mitochondrial membrane depolarization and ATP hydrolysis. Mitochondrial swelling is the major factor leading to mitochondria-mediated cell death through both apoptosis and necrosis. Hence, precise estimation of the threshold parameters of the transition of reversible swelling to irreversible swelling is important for understanding the mechanisms of PTP-mediated cell death as well as for the development of new therapeutic approaches targeting the mitochondria under pathological conditions. In this study, we designed a simple kinetic model of the Ca2+ -induced mitochondrial swelling that describes the mechanisms of transition from reversible to irreversible swelling in cardiac mitochondria. Values of kinetic parameters calculated using parameter estimation techniques that fit experimental data of mitochondrial swelling with minimum average differences between the experimental data and model parameters. Overall, this study provides a kinetic model verified by data simulation and model fitting that adequately describes the dynamics of mitochondrial swelling.

Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction.

The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Genetic deletion of the adaptor protein p66Shc increases susceptibility to short-term ischaemic myocardial injury via intracellular salvage pathways.

AIMS: Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion. METHODS AND RESULTS: Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT. CONCLUSIONS: Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients.

Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria.

Because of the lack of appropriate animal models, the potentially causal contributions of inherited mitochondrial genomic factors to complex traits are less well studied compared with inherited nuclear genomic factors. We previously detected variations between the mitochondrial DNA (mtDNA) of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). Specifically, multiple variations were detected in mitochondrial genes coding for subunits of proteins essential for electron transport, in mitochondrial reactive oxygen species production, and within the D-loop region. To evaluate the effects of these mtDNA variations in the absence of the corresponding nuclear genomic factors as confounding variables, novel reciprocal strains of S and SHR were constructed and characterized. When compared with that of the S rat, the heart tissue from the S.SHR(mt) conplastic strain wherein the mtDNA of the S rat was substituted with that of the SHR had a significant increase in mtDNA copy number and decrease in mitochondrial reactive oxygen species production. A corresponding increase in aerobic treadmill running capacity and a significant increase in survival that was not related to changes in blood pressure were observed in the S.SHR(mt) rats compared with the S rat. The reciprocal SHR.S(mt) rats did not differ from the SHR in any phenotype tested, suggesting lower penetrance of the S mtDNA on the nuclear genomic background of the SHR. These novel conplastic strains serve as invaluable tools to further dissect the relationship between heart function, aerobic fitness, cardiovascular disease progression, and mortality.

Paciente con tumefacción en la articulación esternoclavicular. Síndrome SAPHO / Patient with numbness in the sternoclavicular joint: SAPHO Syndrome

El síndrome SAPHO está constituido por la asociación de alteraciones musculoesqueléticas y alteraciones dermatológicas. Presentamos el caso de un varón de 35 años que cumple criterios de síndrome SAPHO. Aunque el tratamiento de estos pacientes aún no está claro, es importante hacer el diagnóstico del síndrome SAPHO para realizar las investigaciones necesarias e instaurar el tratamiento. El término es un acrónimo de las manifestaciones más frecuentes: sinovitis, acné, pustulosis palmo-plantar, hiperostosis y osteítis (AU)

The SAPHO syndrome describes an association between musculoskeletal disorders and various dermatological conditions. We report the case of a 35-years-old man who fulfilled the criteria for SAPHO. Although the optimal treatment for these patients remains unclear, it is important to make the diagnosis of SAPHO to avoid unnecessary investigations and treatment. SAPHO is an acronym of the combination of synovitis, acne, pustulosis, hyperostosis, and osteitis (AU)

Sod2 knockdown in the musculature has whole-organism consequences in Drosophila.

Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseases and aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxide dismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotor function, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knockdown in the musculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observed with knockdown of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to these phenotypes. Knockdown of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) and caused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content, and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophila and that the consequences of SOD2 loss in this tissue extend to the viability of the organism as a whole.

Mitochondrial alterations in livers of Sod1-/- mice fed alcohol.

Chronic alcohol consumption induced liver injury in Cu,Zn-superoxide dismutase-deficient mice (Sod1-/-), with extensive centrilobular necrosis and inflammation and a reduction in hepatic ATP content. Mechanisms by which ethanol decreased ATP in these mice remain unclear. We investigated alterations in mitochondria of Sod1-/- mice produced by chronic ethanol treatment. These mitochondria had an increase in State 4 oxygen consumption with succinate and especially with glutamate plus malate compared to mitochondria from pair-fed Sod1-/- mice or mitochondria from wild-type mice fed dextrose or ethanol. This uncoupling was associated with a decrease in ADP/O and respiratory control ratios, a decline in mitochondrial membrane potential, enhanced mitochondrial permeability transition, and decreased aconitase activity. Total thiols and uncoupling protein 2 levels were elevated in the pair-fed Sod1-/- mitochondria, perhaps an adaptive response to oxidant stress. However, no such increases were found with the ethanol-fed Sod1-/- mitochondria, suggesting a failure to develop these adaptations. The mitochondria from the ethanol-fed Sod1-/- mice had elevated levels of cleaved Bax, Bak, Bcl-xl, and adenine nucleotide translocator. Immunoprecipitation studies revealed increased association of Bax and Bak with the adenine nucleotide translocator. ADP-ATP exchange was very low in the ethanol-fed Sod1-/- mitochondria. These results suggest that ethanol treatment of Sod1-/- mice produces uncoupling and a decline in Deltapsi, swelling, increased association of proapoptotic proteins involved in the permeability transition, and decreased adenine nucleotide translocator activity, which may be responsible for the decline in ATP levels and development of necrosis in this model of alcohol-induced liver injury.

Pancreatitis associated with hyperlipoproteinaemia type I in mink (Mustela vison): earliest detectable changes occur in mitochondria of exocrine cells.

Pancreatic tissue from young mink homozygous for a mutation in the lipoprotein lipase gene was studied by light and electron microscopy, with the aim of describing the earliest detectable changes in a process which rapidly progresses into overt pancreatitis. The mutation leads to hyperlipoproteinaemia, corresponding to hyperlipoproteinaemia type I in man. Assessment of relevant hepatic and pancreatic enzymes were included in the investigation. The earliest detectable changes consisted of widespread swelling and vacuolation of exocrine cells, arising mainly from swollen mitochondria. To a lesser extent, vesiculation of endoplasmic reticulum occurred. Mitochondria exhibited various changes, including cavitation and dilution of the matrix, with shortened and disorganized cristae displaced towards the periphery. Lamellar figures that developed within mitochondria were numerous. Acinar lumina were somewhat dilated, while plasma membranes were relatively well preserved and secretory granules seemed unchanged. Exfoliative processes progressively occurred, resulting in total necrosis of groups of parenchymal cells, while intercalated ducts were spared. The necrosis was rapidly followed by inflammatory reactions. The activity of the mitochondrial enzyme carnitine O-palmitoyltransferase, essential for the transport of fatty acids into the mitochondria, was lower in the pancreas than in the liver. The activity of the peroxisomal fatty acid beta-oxidation was high in the liver and low in the pancreas of both lipoprotein lipase-deficient and control mink. It is concluded that pancreatic lesions associated with hyperlipoproteinaemia start in exocrine cells, and are most probably the result of a metabolic disturbance, possibly a toxic effect of an excess of free fatty acids.

A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis.

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.

Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.

Mutations in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation sites of the beta-catenin gene exon 3 are found in 20-30% of human primary hepatocellular carcinoma (HCC), whereas mutations in the APC or AXIN genes are found in other HCC populations. These data strongly suggest that the Wnt signaling pathway is involved in hepatocarcinogenesis. To determine the role of beta-catenin in intestinal tumorigenesis, we earlier constructed a mutant mouse strain Catnb(lox(ex3)), in which exon 3 of the beta-catenin gene was sandwiched by loxP sequences. By genetic crosses of these mice with the Fabpl-cre transgenic mice that express the cre gene controlled by the fatty acid binding protein gene promoter, we introduced the beta-catenin stabilizing mutation into the small intestine and liver. Although numerous polyps were formed in the small intestine, we did not find any neoplastic (i.e., dysplastic) foci in the liver, and the mice died in 5 weeks after birth because of acute liver damage accompanying mitochondrial swelling. When a recombinant adenovirus that expresses the cre gene from a human cytomegalovirus early gene promoter was constructed and inoculated at a high multiplicity (10(9) plaque-forming units/mouse), the Catnb(lox(ex3)) mice showed marked hepatomegaly, with similar mitochondrial swelling in the hepatocytes, and died within 3 weeks after infection. On the other hand, when inoculated at lower multiplicities of infection (10(7) and 10(8) plaque-forming units/mouse, respectively), the Catnb(lox(ex3)) mice survived >6 months without any neoplastic foci in the liver, although the nuclear localization of beta-catenin was found in some hepatocytes even after 6 months. These results suggest that, in contrast to intestinal polyposis, the Wnt pathway activation by stabilized beta-catenin is not sufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required.

The role of creatine kinase in inhibition of mitochondrial permeability transition.

Cyclosporin A sensitive swelling of mitochondria isolated from control mouse livers and from the livers of transgenic mice expressing human ubiquitous mitochondrial creatine kinase occurred in the presence of both 40 microM calcium and 5 microM atractyloside which was accompanied by a 2.5-fold increase over state 4 respiration rates. Creatine and cyclocreatine inhibited the latter only in transgenic liver mitochondria. Protein complexes isolated from detergent solubilised rat brain extracts, containing octameric mitochondrial creatine kinase, porin and the adenine nucleotide translocator, were reconstituted into malate loaded lipid vesicles. Dimerisation of creatine kinase in the complexes and exposure of the reconstituted complexes to >200 microM calcium induced a cyclosporin A sensitive malate release. No malate release occurred with complexes containing octameric creatine kinase under the same conditions.