- Email: [email protected]
Spontaneous elimination of mitochondrial mutations during the induction of pluripotency
Abstracts
10 Low level of MtDNA mutation promotes mitochondrial bioenergetics and oxidative metabolism via retrograde signaling Presenter: Sarika Srivastava Sarika Srivastavaa, Karina N. Gonzalez Herreraa, Vincent Proaccciob, Douglas C. Wallacec, Marcia C. Haigisa a Department of Cell Biology, Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA 02115, USA b Department of Biochemistry and Genetics, Angers University Hospital, School of Medicine, and UMR INSERM, U771-CNRS6214, Angers, France c Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA Point mutations in mitochondrial tRNA genes have been associated with mitochondrial encephalomyopathies. The mitochondrial 3243ANG tRNA Leu(UUR) gene mutation exceeding a critical threshold (N85%) leads to a clinical onset of MELAS disease. Below the critical threshold, the mitochondrial DNA (mtDNA) mutation load is clinically asymptomatic with no deleterious impact on cellular bioenergetics. In this study, we report that the low level of m3243ANG mutation activates mitochondrial bioenergetics, biogenesis and cellular metabolism in the MELAS 28% heteroplasmic cybrid (M28) cells compared to the 143B wild-type control cells. Strikingly, we found that the rate of mitochondrial respiration, oxidative capacity, enzyme complex activities and mtDNA levels was significantly higher in the M28 cybrid cells relative to the 143B control cells. The microarray data analysis revealed increased expression of several genes involved in oxidative phosphorylation, TCA cycle and fatty-acid metabolism pathways in the M28 cybrid cells relative to the 143B control. Mechanistically, we found that multiple transcription factors and coactivators involved in regulating mitochondrial gene expression and lipid metabolism were markedly stimulated in M28 cybrid cells relative to the 143B control cells. Furthermore, the energy and nutrient sensing AMPK signaling pathway was significantly activated in the M28 cybrid cells relative to the 143B control cells. Our findings suggest that the low level of mtDNA mutation is a potential signal for mitochondrial dysfunction and that the nuclear genome apparatus senses and responds via retrograde signaling which consequently leads to the activation of the transcriptional regulatory network to further enhance mitochondrial bioenergetics, biogenesis and metabolism. This overall positive impact of the low level of mtDNA mutation on mitochondrial function implicates that the pharmacological modulation of the underlying signaling pathways may boost mitochondrial bioenergetics and opens new avenues for mitochondrial disease therapeutics.
doi:10.1016/j.mito.2012.07.009
11 Rapid breath test for in vivo determination of human pyruvate dehydrogenase complex activity Presenter: Peter W. Stacpoole, Ph.D., M.D. Peter W. Stacpoolea, David A. Wagnerb a University of Florida College of Medicine, Division of Endocrinology, Diabetes and Metabolism, Gainesville, FL, USA b Metabolic Solutions, Inc., Nashua, NH, USA Abstract: The pyruvate dehydrogenase complex (PDC) is a key regulatory enzyme in cellular energy metabolism. Under aerobic conditions, PDC catalyzes the rate-determining step in glucose oxidation by irreversibly decarboxylating pyruvate to acetyl CoA and CO2. In this way, the PDC links glycolysis with the citric acid cycle and gluconeogenesis, as well as both lipid and amino acid metabolism.
553
Regulation of the PDC is achieved by reversible phosphorylation by families of pyruvate dehydrogenase kinases (PDKs) and phosphatases (PDPs), in which the phosphorylated form of the PDC is inactive. Prior human research on PDC regulation has mainly employed in vitro assays of isolated human cells or invasive skeletal muscle biopsies. However, these approaches fail to provide a safe and facile means of serially assessing whole body PDC activity. We have addressed these shortcomings by developing the Pyruvate Breath Test (PBT) that uses a small, oral dose of sodium 1-13C-pyruvate to determine PDC activity based on the conversion of 13C-pyruvate to 13CO2. A pilot study in two subjects was conducted to provide proof-ofconcept for the use of an oral PBT as a tool for assessing mitochondrial activity of the PDC and in response to therapeutic intervention using dichloroacetate (DCA), a prototypic PDK inhibitor. 13CO2 production in exhaled air was measured from an oral 100 mg dose of 1-13C-pyruvate in the basal, overnight fasted state and, after one-week washout, again in the fasted state 1 h after oral administration of 25 mg/kg of DCA. Oral 13C-pyruvate administration resulted in a typical concentration vs. time-dependent curve, with 13CO2 detectable within 5 min of dosing, reaching maximum levels in 20–40 min and decreasing gradually over 2+ hours. In both subjects, DCA exposure rapidly stimulated PDC activity during the initial 30 min of breath collections, as evidenced by the upward and leftward shift in the concentration– time curve. We determined in one subject whether a lower oral 113 C-pyruvate dose could be utilized. Cumulative % recovery of the dose as 13CO2 over 30 min was the same following doses of 25, 50 and 100 mg of 1-13C-pyruvate, indicating that an oral dose of 25 mg could be employed in future studies, using fewer samples and a shorter duration. In summary, these data suggest that the PBT may provide a safe and rapid in vivo measure of a dynamic and critically important mitochondrial bioenergetic reaction within 30 min of oral substrate administration and can be performed serially under physiological, pharmacological and pathological conditions. doi:10.1016/j.mito.2012.07.010
12 Spontaneous elimination of mitochondrial mutations during the induction of pluripotency Presenter: Neal Sondheimer Neal Sondheimera, Ornella Zolloa, Jason A. Millsb, Catherine E. Glatza, Paul Gadueb, Deborah L. Frenchb a Division of Child Rehabilitation and Biochemical Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States b Center For Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States Introduction: The correction of genetic lesions in pluripotent stem cells represents an important first step in the development of cell-based therapies. Disorders due to mitochondrial DNA (mtDNA) mutations present an intriguing target for cell-based therapy because of unique features of mitochondrial genetics. mtDNA mutations are often heteroplasmic, where both wild-type and mutant sequences are present at the level of the organism, tissue or cell. The effect of these mutations is often determined by the level or load of heteroplasmy, and the reduction of pathological heteroplasmy is an important goal of mitochondrial therapies. The objective of this study was to determine the dynamics of mitochondrial heteroplasmy during the conversion of fibroblasts to induced pluripotent stem cells (iPSCs). Methods: We generated iPSCs from four patients with heteroplasmic disease-causing mutations, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and three with Leigh syndrome
554
Abstracts
and one unaffected infant. Mitochondrial function in iPSCs was assessed by fluorescence-associated cell sorting analysis of Mito Tracker CMXRos straining and oxygen consumption. Results: The generated cell lines fulfilled criteria for pluripotency, including the expression of pluripotency markers and the ability to form teratomas in mice. An evaluation of mitochondrial genotype demonstrated the spontaneous elimination of heteroplasmy. The iPSC lines generated were homoplasmic, with mutations either entirely eliminated or with no remaining wild-type DNA. Generation of iPSCs from a cell line with non-pathogenic heteroplasmy gave a similar result, suggesting that this result was not due to any selection for or against pathogenic mutations. Studies of the cells from which iPSCs were derived suggested that the bottleneck could be due to the presence of homoplasmic fibroblast clones within an otherwise mixed population. Functional studies of mitochondrial activity demonstrated that mitochondrial membrane potential was impaired in iPSCs bearing pathogenic mutations. Conclusions: Our ability to generate homoplasmic, pluripotent cells from patients with disease may be a mechanism for eliminating disease-causing mutations in cells with future therapeutic potential.
diabetes) show increased glucose production and gluconeogenesis rates when compared to the control subjects. Diabetic subjects with MELAS exhibit higher insulin resistance as calculated by HOMA, whereas non-diabetic subjects with MELAS show a higher rate of glucose clearance. Conclusions: This interim analysis reveals that subjects with MELAS syndrome have abnormalities in glucose metabolism. Subjects with MELAS who do not have DM have higher rates of glucose production and gluconeogenesis that can predispose them to develop diabetes. Subjects with MELAS and diabetes showed both increased glucose production and higher insulin resistance, suggesting that DM develops due to multiple defects in glucose metabolism in MELAS. The completion of this study will result in a better understanding of the pathophysiological mechanisms of DM in subjects with MELAS syndrome, which can influence the management and prognosis of the disorder and may provide further insights into the pathogenesis of DM in mitochondrial diseases in general.
doi:10.1016/j.mito.2012.07.011
14
doi:10.1016/j.mito.2012.07.012
The role of a taurine-containing wobble modification deficiency 13 Glucose kinetics in subjects with MELAS syndrome: Interim results Presenter: Lisa Emrick Ayman W. El-Hattaba, Lisa Emrickb, Jean W.C. Hsuc, Farook Jahoorc, Fernando Scagliab, William Craigenb a Division of Medical Genetics, Department of Child Health, University of Missouri Health Care, Columbia, MO, United States b Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States c US Department of Agriculture/Agricultural Research Service—Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas, United States Background: The mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders in which diabetes mellitus (DM) occurs in one third of affected individuals. The pathogenesis of DM in MELAS syndrome remains unclear. We hypothesize that DM develops in individuals with MELAS syndrome due to multiple defects in glucose metabolism, including decreased glucose utilization, increased glucose production, decreased insulin secretion, and increased insulin resistance. Individuals with MELAS syndromes who do not yet have DM may have altered glucose metabolism. Methods: In this study we aim to measure the rates of endogenous glucose production, gluconeogenesis, glucose oxidation, and glucose clearance via stable isotope infusion technique in subjects with MELAS syndrome who have DM, subjects with MELAS syndrome who do not have DM, and in healthy control subjects. In addition, we measure the concentrations of fasting blood glucose, insulin, and glycosylated hemoglobin (HbA1c); and assess insulin resistance using Homeostatic Model Assessment (HOMA). The research subjects are admitted to the General Clinical Research Center at Texas Children's Hospital. After a 12 hour-fast, the isotope infusion is started with a priming dose of NaH13CO3 and U-13C6 glucose followed by continuous infusion of U-13C6 glucose for 6 h. Blood and breath samples are collected and analyzed for isotopic enrichments. Results: To date, 6 control subjects, 4 subjects with MELAS and DM, and 4 non-diabetic subjects with MELAS have completed the study. Both groups of subjects with MELAS (with and without
in MELAS Presenter: Chian Ju Jong Chian Ju Jonga, K.C. Ramilaa, Takashi Itob, Junichi Azumab, Stephen Schaffera a University of South Alabama, Department of Pharmacology, College of Medicine, Mobile, AL 36688, United States b Hyogo University of Health Sciences, School of Pharmacy, Kobe, Japan MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease that usually coexists with a cardiomyopathy. The most common pathogenic mutation in MELAS is an A to G transition at position 3243 in the aminoacyl stem of mitochondrial tRNALeu(UUR). The A3243G mutation affects tRNALeu(UUR) structure and stability, aminoacylation and posttranscriptional modification of a wobble base, all of which, presumably, lead to a decrease in the synthesis of mitochondrial proteins. However, it is unclear if wobble modification deficiency causes MELAS-like changes in the respiratory chain that lead to cardiac contractile dysfunction. To examine the effect of wobble modification deficiency in mitochondrial function, we developed a mouse model lacking taurine transporter (TauTKO), which diminishes the levels of a substrate (taurine) required for the conversion of uridine to 5taurinomethyluridine at the wobble position of tRNALeu(UUR). We showed that TauTKO hearts demonstrated decreased levels of several complex I subunits, reduced complex I activity and suppressed oxygen consumption. There was also evidence of oxidative stress, as exemplified by a decrease in both aconitase activity and glutathione redox ratio, and an increase in MitoSox fluorescence. We also showed activation of protein kinase C (PKC)-δ and PKC-ε and an increase in the phosphorylation state of troponin I, which regulates contractile function. Taurine depletion was also associated with development of cardiac dysfunction, as characterized by a decrease in both fractional shortening and ejection fraction, a decline in Ca2 +-dependent myofibrillar ATPase activity and an increase in mRNA levels of heart failure markers such as ANP, BNP and β-MHC genes. Our findings suggest that a wobble defect leads to impaired respiratory chain activity, resulting in oxidative stress. This is consistent with the idea that a wobble defect decreases the efficiency of UUG decoding for synthesis of mitochondrial proteins. This leads to a defect in respiratory chain flux and a diversion of electrons from the respiratory chain to the acceptor, oxygen, forming in the process superoxide. Oxidative stress is a potential trigger of contractile
10 Low level of MtDNA mutation promotes mitochondrial bioenergetics and oxidative metabolism via retrograde signaling Presenter: Sarika Srivastava Sarika Srivastavaa, Karina N. Gonzalez Herreraa, Vincent Proaccciob, Douglas C. Wallacec, Marcia C. Haigisa a Department of Cell Biology, Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA 02115, USA b Department of Biochemistry and Genetics, Angers University Hospital, School of Medicine, and UMR INSERM, U771-CNRS6214, Angers, France c Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA Point mutations in mitochondrial tRNA genes have been associated with mitochondrial encephalomyopathies. The mitochondrial 3243ANG tRNA Leu(UUR) gene mutation exceeding a critical threshold (N85%) leads to a clinical onset of MELAS disease. Below the critical threshold, the mitochondrial DNA (mtDNA) mutation load is clinically asymptomatic with no deleterious impact on cellular bioenergetics. In this study, we report that the low level of m3243ANG mutation activates mitochondrial bioenergetics, biogenesis and cellular metabolism in the MELAS 28% heteroplasmic cybrid (M28) cells compared to the 143B wild-type control cells. Strikingly, we found that the rate of mitochondrial respiration, oxidative capacity, enzyme complex activities and mtDNA levels was significantly higher in the M28 cybrid cells relative to the 143B control cells. The microarray data analysis revealed increased expression of several genes involved in oxidative phosphorylation, TCA cycle and fatty-acid metabolism pathways in the M28 cybrid cells relative to the 143B control. Mechanistically, we found that multiple transcription factors and coactivators involved in regulating mitochondrial gene expression and lipid metabolism were markedly stimulated in M28 cybrid cells relative to the 143B control cells. Furthermore, the energy and nutrient sensing AMPK signaling pathway was significantly activated in the M28 cybrid cells relative to the 143B control cells. Our findings suggest that the low level of mtDNA mutation is a potential signal for mitochondrial dysfunction and that the nuclear genome apparatus senses and responds via retrograde signaling which consequently leads to the activation of the transcriptional regulatory network to further enhance mitochondrial bioenergetics, biogenesis and metabolism. This overall positive impact of the low level of mtDNA mutation on mitochondrial function implicates that the pharmacological modulation of the underlying signaling pathways may boost mitochondrial bioenergetics and opens new avenues for mitochondrial disease therapeutics.
doi:10.1016/j.mito.2012.07.009
11 Rapid breath test for in vivo determination of human pyruvate dehydrogenase complex activity Presenter: Peter W. Stacpoole, Ph.D., M.D. Peter W. Stacpoolea, David A. Wagnerb a University of Florida College of Medicine, Division of Endocrinology, Diabetes and Metabolism, Gainesville, FL, USA b Metabolic Solutions, Inc., Nashua, NH, USA Abstract: The pyruvate dehydrogenase complex (PDC) is a key regulatory enzyme in cellular energy metabolism. Under aerobic conditions, PDC catalyzes the rate-determining step in glucose oxidation by irreversibly decarboxylating pyruvate to acetyl CoA and CO2. In this way, the PDC links glycolysis with the citric acid cycle and gluconeogenesis, as well as both lipid and amino acid metabolism.
553
Regulation of the PDC is achieved by reversible phosphorylation by families of pyruvate dehydrogenase kinases (PDKs) and phosphatases (PDPs), in which the phosphorylated form of the PDC is inactive. Prior human research on PDC regulation has mainly employed in vitro assays of isolated human cells or invasive skeletal muscle biopsies. However, these approaches fail to provide a safe and facile means of serially assessing whole body PDC activity. We have addressed these shortcomings by developing the Pyruvate Breath Test (PBT) that uses a small, oral dose of sodium 1-13C-pyruvate to determine PDC activity based on the conversion of 13C-pyruvate to 13CO2. A pilot study in two subjects was conducted to provide proof-ofconcept for the use of an oral PBT as a tool for assessing mitochondrial activity of the PDC and in response to therapeutic intervention using dichloroacetate (DCA), a prototypic PDK inhibitor. 13CO2 production in exhaled air was measured from an oral 100 mg dose of 1-13C-pyruvate in the basal, overnight fasted state and, after one-week washout, again in the fasted state 1 h after oral administration of 25 mg/kg of DCA. Oral 13C-pyruvate administration resulted in a typical concentration vs. time-dependent curve, with 13CO2 detectable within 5 min of dosing, reaching maximum levels in 20–40 min and decreasing gradually over 2+ hours. In both subjects, DCA exposure rapidly stimulated PDC activity during the initial 30 min of breath collections, as evidenced by the upward and leftward shift in the concentration– time curve. We determined in one subject whether a lower oral 113 C-pyruvate dose could be utilized. Cumulative % recovery of the dose as 13CO2 over 30 min was the same following doses of 25, 50 and 100 mg of 1-13C-pyruvate, indicating that an oral dose of 25 mg could be employed in future studies, using fewer samples and a shorter duration. In summary, these data suggest that the PBT may provide a safe and rapid in vivo measure of a dynamic and critically important mitochondrial bioenergetic reaction within 30 min of oral substrate administration and can be performed serially under physiological, pharmacological and pathological conditions. doi:10.1016/j.mito.2012.07.010
12 Spontaneous elimination of mitochondrial mutations during the induction of pluripotency Presenter: Neal Sondheimer Neal Sondheimera, Ornella Zolloa, Jason A. Millsb, Catherine E. Glatza, Paul Gadueb, Deborah L. Frenchb a Division of Child Rehabilitation and Biochemical Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States b Center For Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States Introduction: The correction of genetic lesions in pluripotent stem cells represents an important first step in the development of cell-based therapies. Disorders due to mitochondrial DNA (mtDNA) mutations present an intriguing target for cell-based therapy because of unique features of mitochondrial genetics. mtDNA mutations are often heteroplasmic, where both wild-type and mutant sequences are present at the level of the organism, tissue or cell. The effect of these mutations is often determined by the level or load of heteroplasmy, and the reduction of pathological heteroplasmy is an important goal of mitochondrial therapies. The objective of this study was to determine the dynamics of mitochondrial heteroplasmy during the conversion of fibroblasts to induced pluripotent stem cells (iPSCs). Methods: We generated iPSCs from four patients with heteroplasmic disease-causing mutations, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and three with Leigh syndrome
554
Abstracts
and one unaffected infant. Mitochondrial function in iPSCs was assessed by fluorescence-associated cell sorting analysis of Mito Tracker CMXRos straining and oxygen consumption. Results: The generated cell lines fulfilled criteria for pluripotency, including the expression of pluripotency markers and the ability to form teratomas in mice. An evaluation of mitochondrial genotype demonstrated the spontaneous elimination of heteroplasmy. The iPSC lines generated were homoplasmic, with mutations either entirely eliminated or with no remaining wild-type DNA. Generation of iPSCs from a cell line with non-pathogenic heteroplasmy gave a similar result, suggesting that this result was not due to any selection for or against pathogenic mutations. Studies of the cells from which iPSCs were derived suggested that the bottleneck could be due to the presence of homoplasmic fibroblast clones within an otherwise mixed population. Functional studies of mitochondrial activity demonstrated that mitochondrial membrane potential was impaired in iPSCs bearing pathogenic mutations. Conclusions: Our ability to generate homoplasmic, pluripotent cells from patients with disease may be a mechanism for eliminating disease-causing mutations in cells with future therapeutic potential.
diabetes) show increased glucose production and gluconeogenesis rates when compared to the control subjects. Diabetic subjects with MELAS exhibit higher insulin resistance as calculated by HOMA, whereas non-diabetic subjects with MELAS show a higher rate of glucose clearance. Conclusions: This interim analysis reveals that subjects with MELAS syndrome have abnormalities in glucose metabolism. Subjects with MELAS who do not have DM have higher rates of glucose production and gluconeogenesis that can predispose them to develop diabetes. Subjects with MELAS and diabetes showed both increased glucose production and higher insulin resistance, suggesting that DM develops due to multiple defects in glucose metabolism in MELAS. The completion of this study will result in a better understanding of the pathophysiological mechanisms of DM in subjects with MELAS syndrome, which can influence the management and prognosis of the disorder and may provide further insights into the pathogenesis of DM in mitochondrial diseases in general.
doi:10.1016/j.mito.2012.07.011
14
doi:10.1016/j.mito.2012.07.012
The role of a taurine-containing wobble modification deficiency 13 Glucose kinetics in subjects with MELAS syndrome: Interim results Presenter: Lisa Emrick Ayman W. El-Hattaba, Lisa Emrickb, Jean W.C. Hsuc, Farook Jahoorc, Fernando Scagliab, William Craigenb a Division of Medical Genetics, Department of Child Health, University of Missouri Health Care, Columbia, MO, United States b Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States c US Department of Agriculture/Agricultural Research Service—Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas, United States Background: The mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders in which diabetes mellitus (DM) occurs in one third of affected individuals. The pathogenesis of DM in MELAS syndrome remains unclear. We hypothesize that DM develops in individuals with MELAS syndrome due to multiple defects in glucose metabolism, including decreased glucose utilization, increased glucose production, decreased insulin secretion, and increased insulin resistance. Individuals with MELAS syndromes who do not yet have DM may have altered glucose metabolism. Methods: In this study we aim to measure the rates of endogenous glucose production, gluconeogenesis, glucose oxidation, and glucose clearance via stable isotope infusion technique in subjects with MELAS syndrome who have DM, subjects with MELAS syndrome who do not have DM, and in healthy control subjects. In addition, we measure the concentrations of fasting blood glucose, insulin, and glycosylated hemoglobin (HbA1c); and assess insulin resistance using Homeostatic Model Assessment (HOMA). The research subjects are admitted to the General Clinical Research Center at Texas Children's Hospital. After a 12 hour-fast, the isotope infusion is started with a priming dose of NaH13CO3 and U-13C6 glucose followed by continuous infusion of U-13C6 glucose for 6 h. Blood and breath samples are collected and analyzed for isotopic enrichments. Results: To date, 6 control subjects, 4 subjects with MELAS and DM, and 4 non-diabetic subjects with MELAS have completed the study. Both groups of subjects with MELAS (with and without
in MELAS Presenter: Chian Ju Jong Chian Ju Jonga, K.C. Ramilaa, Takashi Itob, Junichi Azumab, Stephen Schaffera a University of South Alabama, Department of Pharmacology, College of Medicine, Mobile, AL 36688, United States b Hyogo University of Health Sciences, School of Pharmacy, Kobe, Japan MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease that usually coexists with a cardiomyopathy. The most common pathogenic mutation in MELAS is an A to G transition at position 3243 in the aminoacyl stem of mitochondrial tRNALeu(UUR). The A3243G mutation affects tRNALeu(UUR) structure and stability, aminoacylation and posttranscriptional modification of a wobble base, all of which, presumably, lead to a decrease in the synthesis of mitochondrial proteins. However, it is unclear if wobble modification deficiency causes MELAS-like changes in the respiratory chain that lead to cardiac contractile dysfunction. To examine the effect of wobble modification deficiency in mitochondrial function, we developed a mouse model lacking taurine transporter (TauTKO), which diminishes the levels of a substrate (taurine) required for the conversion of uridine to 5taurinomethyluridine at the wobble position of tRNALeu(UUR). We showed that TauTKO hearts demonstrated decreased levels of several complex I subunits, reduced complex I activity and suppressed oxygen consumption. There was also evidence of oxidative stress, as exemplified by a decrease in both aconitase activity and glutathione redox ratio, and an increase in MitoSox fluorescence. We also showed activation of protein kinase C (PKC)-δ and PKC-ε and an increase in the phosphorylation state of troponin I, which regulates contractile function. Taurine depletion was also associated with development of cardiac dysfunction, as characterized by a decrease in both fractional shortening and ejection fraction, a decline in Ca2 +-dependent myofibrillar ATPase activity and an increase in mRNA levels of heart failure markers such as ANP, BNP and β-MHC genes. Our findings suggest that a wobble defect leads to impaired respiratory chain activity, resulting in oxidative stress. This is consistent with the idea that a wobble defect decreases the efficiency of UUG decoding for synthesis of mitochondrial proteins. This leads to a defect in respiratory chain flux and a diversion of electrons from the respiratory chain to the acceptor, oxygen, forming in the process superoxide. Oxidative stress is a potential trigger of contractile