Next-generation sequencing for mitochondrial disorders.

A great deal of our understanding of mitochondrial function has come from studies of inherited mitochondrial diseases, but still majority of the patients lack molecular diagnosis. Furthermore, effective treatments for mitochondrial disorders do not exist. Development of therapies has been complicated by the fact that the diseases are extremely heterogeneous, and collecting large enough cohorts of similarly affected individuals to assess new therapies properly has been difficult. Next-generation sequencing technologies have in the last few years been shown to be an effective method for the genetic diagnosis of inherited mitochondrial diseases. Here we review the strategies and findings from studies applying next-generation sequencing methods for the genetic diagnosis of mitochondrial disorders. Detailed knowledge of molecular causes also enables collection of homogenous cohorts of patients for therapy trials, and therefore boosts development of intervention.

Cardioprotection mediated by urocortin is dependent on PKCepsilon activation.

Urocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. We have found that the mechanism of action of Ucn involves both acute activation of specific target molecules, and using Affymetrix (Santa Clara, CA) gene chip technology, altered gene expression of different end effector molecules. Here, from our gene chip data, we show that after a 24 h exposure to Ucn, there was a specific increase in mRNA and protein levels of the protein kinase C epsilon (PKCepsilon) isozyme in primary rat cardiomyocytes compared with untreated cells and in the Langendorff perfused ex vivo heart. Furthermore, a short 10 min exposure of these cells to Ucn caused a specific translocation/activation of PKCepsilon in vitro and in the Langendorff perfused ex vivo heart. The importance of the PKCepsilon isozyme in cardioprotection and its relationship to cardioprotection produced by Ucn was assessed using PKCepsilon-specific inhibitor peptides. The inhibitor peptide, when introduced into cardiomyocytes, caused an increase in apoptotic cell death compared with control peptide after ischemia and reperfusion. When the inhibitor peptide was present with Ucn, the cardioprotective effect of Ucn was lost. This loss of cardioprotection by Ucn was also seen in whole hearts from PKCepsilon knockout mice. These findings indicate that the cardioprotective effect of Ucn is dependent upon PKCepsilon.

Signal-transduction pathways involved in the hypertrophic effect of hsp56 in neonatal cardiomyocytes.

Heat shock protein (hsp) 56 (hsp56) is an immunophilin that acts as a cofactor with hsp90 and exhibits both peptidyl-prolyl isomerase activity and chaperone activity. Previous studies have shown that the hypertrophic effect of cardiotrophin-1 (CT-1) in cardiac cells is dependent on hsp56 induction. CT-1 activates a number of signal-transduction pathways. Therefore, we sought to determine whether these pathways were also important for hsp56-induced hypertrophy using overexpression with transiently transfected plasmid vectors in rat neonatal cardiomyocytes. Here we show that multiple signalling pathways are involved in hsp56-induced hypertrophy, namely the Janus kinase-signal-transducer and activator of transcription, extracellular signal-regulated protein kinase and PI3-kinase/Akt signalling pathways. Dominant-negative mutants and inhibitors of these pathways were able to block the hypertrophy observed as a result of hsp56 overexpression. However, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway was not able to block the hypertrophic effect of hsp56 overexpression. Furthermore, we show that domains I, II and IV of the hsp56 protein may be involved in its hypertrophic effect. These studies show for the first time that multiple signalling pathways are involved in the hypertrophic effect of hsp56 and that overexpression of hsp56 itself is able to activate the necessary signalling pathways, which induce hypertrophy.

Urocortin protects cardiac myocytes from ischemia/reperfusion injury by attenuating calcium-insensitive phospholipase A2 gene expression.

We have used Affymetrix gene chip technology to look for changes in gene expression caused by a 24 h exposure of rat primary neonatal cardiac myocytes to the cardioprotective agent urocortin. We observed a 2.5-fold down-regulation at both the mRNA and protein levels of a specific calcium-insensitive phospholipase A2 enzyme. Levels of lysophosphatidylcholine, a toxic metabolite of phospholipase A2, were lowered by 30% in myocytes treated with urocortin for 24 h and by 50% with the irreversible iPLA2 inhibitor bromoenol lactone compared with controls. Both 4 h ischemia and ischemia followed by 24 h reperfusion caused a significant increase in lysophosphatidylcholine concentration compared with controls. When these myocytes were pretreated with urocortin, the ischemia-induced increase in lysophosphatidylcholine concentration was significantly lowered. Moreover, co-incubation of cardiac myocytes with urocortin, or the specific phospholipase A2 inhibitor bromoenol lactone, reduces the cytotoxicity produced by lysophosphatidylcholine or ischemia/reperfusion. Similarly, in the intact heart ex vivo we found that cardiac damage measured by infarct size was significantly increased when lysophoshatidylcholine was applied during ischemia, compared with ischemia alone, and that pre-treatment with both urocortin and bromoenol lactone reversed the increase in infarct size. This, to our knowledge, is the first study linking the cardioprotective effect of urocortin to a decrease in a specific enzyme protein and a subsequent decrease in the concentration of its cardiotoxic metabolite.

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

Selective induction of labour.

In a prospective study of 1000 consecutive primigravidae labour was induced on 95 occasions. None of 16 perinatal deaths and none of 4 cases of suspected brain damage occurred after prolonged pregnancy or pre-eclampsia. It is concluded that a low incidence of induction is compatible with good results and that enthusiasm for the statistical concept of high risk in obstetric practice should be reviewed in the interest of mothers and children as individuals.