The Biochemical, Molecular Genetics And Pharmacotherapeutic Studies Of Mitochondria-Related Metabolic Myopathy

Metabolic myopathies are inborn errors of metabolism that result in impaired energy production due to defects in glycogen, lipid, mitochondrial, and possibly adenine nucleotide metabolism. Fattyacid oxidation defects (FAOD), glycogen storage disease, and mitochondrial myopathies represent the3main groups of disorders. Clinically, a variety of neuromuscular presentations are seen at different ages of life. Newborns and infants commonly present with hypotonia and multisystem involvement (liver and brain), whereas onset later in life usually presents with exercise intolerance with or without progressive muscle weakness and myoglobinuria. In general, the glycogen storage diseases result in high-intensity exercise intolerance, whereas the FAODs and the mitochondrial myopathies manifest predominately during endurance-type activity or under fasted or other metabolically stressful conditions.In addition to the vast array of mutations found within the maternally inherited mtDNA, there is an increasing recognition of the mitochondrial disorders arising from mutations within the nuclear genome and following Mendelian genetic inheritance patterns. For example, the mutations responsible for a number of autosomal recessive conditions have been found for myoneurogastrointestinal encephalomyopathy (MNGIE, thymidine phosphorylase), complex Ⅰ (NDUF) and Ⅳ (SURF-1) Leigh disease, mtDNA depletion (dGuOK and TK), and some forms of mtDNA deletion syndromes (polymerase gamma, twinkle, adenine nucleotide translocase, and OPA1). Autosomal dominant inheritance patterns have also been seen in rare cases of mtDNA deletion syndromes. Although considered rare, mutations affecting the mitochondrial import system have been described.In this study, we focus on two of the metabolic diseases:One is named multiple coenzyme A dehydrogenation deficiency (MADD) caused by nuclear ETFDH gene mutation, which belongs to fatty acid oxidation defects(FAODs).The gene-encoded protein ETF-QO performs function of electron transfer from beta-oxidation to respirotary chain in mitochondria. We concentrate to reveal the pathogenic mechanism of this disease and therapeutic mechanism of riboflavin with this disease. The other disease, named mitochondrial DNA depletion syndrome (MDS), was caused by nuclear TK2gene mutation. The gene product has lost its major part of function and resulted in down-regulation of mitochondrial dNTP synthesis, leading to multiple defects of mitochondrial DNA encoded proteins. To further study the relationships between mitochondrial activity, reactive oxygen species and calcium handling, we developed an extra study using glibenclamide and macrophages to gain more insight to the mitochondria-related metabolic myopathies.PART I Riboflavin Up-regulates Mitochondrial Flavin Adenine Dinucleotide (FAD) Level Which May Stabilize Flavoprotein in Fibroblasts from Chinese Riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency (RR-MADD) Patients with ETFDH MutationsBackground:Riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency (RR-MADD) has been linked to deficiencies in multiple mitochondrial enzymatic functions for a long time, however, it wasn’t until recently that genetic defects in electron transfer flavoproteins (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) were found to cause RR-MADD. Despite these advances the impact of treatment with riboflavin in human patients with respect to changes in mitochondrial enzyme expression and activity is incompletely understood. Method:The present study uses muscle biopsy and cultured fibroblasts from seven genetically confirmed Chinese RR-MADD patients, to examine the molecular features of both ETF-QO and succinate dehydrogenase (SDH), and changes in mitochondrial FAD concentration, following treatment with riboflavin. Results: Our results show that the primary concentration of mitochondrial FAD was significantly lower in patients than normal controls, and that supplementation of riboflavin partially replenished the mitochondrial FAD concentration in patients. In close correlation with FAD, the protein quantity of ETF-QO was promoted by riboflavin supplementation. A mild depression in succinate dehydrogenase expression and activity could also be rescued by riboflavin supplementation. Conclusion:There exists an abnormal circulating FAD pool, reduced ETF-QO protein and decreased flavoprotein activities in mitochondria of RR-MADD patients which may impact the normal function of other mitochondrial flavoproteins. Supplementation of riboflavin might up-regulate the amount of ETF-QO holoenzymes by producing more FAD that can facilitate binding with apoenzymes.PART II iTRAQ Protein Profile Analysis of muscle with riboflavin-responsive multiple acyl-coenzyme A dehydrogenation deficiency before and after administration of riboflavinMultiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid oxidation disorder caused by mutations that affect electrontransfer flavoprotein (ETF) or ETF:ubiquinone oxidoreductase (ETF-QO) A mild form of MADD characterized by proximal muscle weakness and exercise intolerance showed a significant response to treatment with riboflavin,resulting in near-normalized biochemical and clinical parameters after high doeses of oral riboflavin supplementation. Except for all the available data on the molecular basis of FAO disorders, including MADD, the pathophysiological mechanisms underlying clinical phenotype development are poorly understood. We studied the proteome of two muscle specimen from one case of RR-MADD before and after riboflavin administration, using high-throughput iTRAQ and high-resolution mass spectrometry, and identified44proteins as potential influence-targets of riboflavin. The identified proteins are involved in several pathways including mitochondrial proliferation, glycolysis, β-oxidative of fatty acid and cell death. After analysis and hypothesis with programmed software, we propose a possible model named ’riboflavin switch on-and-off model’, which can explain the switch of metabolism from fatty acid metabolism to glycolysis, as well as a series of changes including FAD pool and stress-induced cell death. Our study provides a global and further perspective of therapeutic mechanism of riboflavin with cases of RR-MADD and highlights the main molecular pathways involved in its pathogenesis.PART Ⅲ A Novel Point Mutation and a Large deletion of TK2Gene Cause Mitochondrial DNA Depletion Syndrome with Infantile Onset of Lipid Storage MyopathyMitochondrial thymidine kinase2(TK2) is a deoxynucleoside kinase that catalyzes the primary phosphorylation of thymidine and deoxycytidine to the corresponding nucleoside monophosphates. In this study, we report an infant with the myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS), harboring a novel heterozygous point mutation (p.R104H) in the TK2gene. The subsequent Multiplex Ligation-dependent Probe Amplification (MLPA) analysis identified a large deletion encompassing exon5to10in the other allele of TK2. The patient developed a progressive myopathy at the age of8months and deceased5months later. Muscle biopsy showed mosaic pattern with COX deficient fibers as well as prominently increased lipid droplets in most muscle fibers. Mitochondrial respiratory chain enzyme activities determined in cultured fibroblasts revealed a moderate decrease of complex II and severe decrease of complex Ⅰ and Ⅳ. The mtDNA copy number in the patient’s muscle and lymphocytes were both markedly decreased. The phenotype characterized by infantile onset and lipid storage in muscle fibers in the present patient extended the clinical and pathological spectrum of MDS caused by TK2deficiency. The large deletion of TK2gene may result in severe dysfunctions of the protein and lead to disturbances of respiratory chains and lipid metabolism.PART IV Glibenclamide Decreases ATP-Induced Intracellular Calcium Transient Elevation via Inhibiting Reactive Oxygen Species and Mitochondrial Activity in MacrophagesIncreasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclearwhether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca2+]i)handling in Raw264.7macrophages. In the present study,[Ca2+]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K+channel blockers had no effect on the resting [Ca2+]i of Raw264.7cells. Extracellular ATP (100mM) induced [Ca2+]i transient elevation independent of extracellular Ca2+. The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca2+]i transient elevation, but pinacidil and other unselective K+channel blockers had no effect. Glibenclamide also decreased the peak of [Ca2+]i transient induced byextracellular thapsigargin (Tg,1mM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone glibenclamide could not decrease ATP, and Tg induced maximal [Ca2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw264.7macrophages.