Published online before print February 23, 2009, doi: 10.1161/CIRCULATIONAHA.108.792101
(Circulation. 2009;119:1272-1283.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Contribution of Impaired Myocardial Insulin Signaling to Mitochondrial Dysfunction and Oxidative Stress in the Heart
From the Division of Endocrinology, Metabolism and Diabetes and Program in Molecular Medicine (S.B., H.B., S.S., B.T.O., V.G.Z., O.I., J.J.W., P.K.M., E.P., T.J.T., H.T., E.D.A.), Department of Biochemistry (O.K., E.D.A.), Division of Cardiology (B.W., S.E.L.), and Department of Family and Preventive Medicine (X.S.), University of Utah School of Medicine, Salt Lake City; Department of Biological Sciences, Idaho State University, Pocatello (K.J.R.); and Center for Integrated BioSystems, Utah State University, Logan (R.C., D.C., B.E.W.).
Correspondence to E. Dale Abel, Division of Endocrinology, Metabolism and Diabetes, Program in Molecular Medicine, 15 N 2030 E Bldg No. 533, Room 3110B, Salt Lake City, Utah 84112. E-mail dale.abel{at}hmbg.utah.edu
Received September 20, 2007; accepted January 6, 2009.
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Abstract |
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Background— Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to left ventricular dysfunction. The contribution of altered myocardial insulin action, independent of associated changes in systemic metabolism, is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function.
Methods and Results— In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced, and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrate glutamate and the fatty acid derivative palmitoyl-carnitine were observed. Mitochondria also were uncoupled when exposed to palmitoyl-carnitine, in part as a result of increased reactive oxygen species production and oxidative stress. Although proteomic and genomic approaches revealed a reduction in subsets of genes and proteins related to oxidative phosphorylation, no reductions in maximal activities of mitochondrial electron transport chain complexes were found. However, a disproportionate reduction in tricarboxylic acid cycle and fatty acid oxidation proteins in mitochondria suggests that defects in fatty acid and pyruvate metabolism and tricarboxylic acid flux may explain the mitochondrial dysfunction observed.
Conclusions— Impaired myocardial insulin signaling promotes oxidative stress and mitochondrial uncoupling, which, together with reduced tricarboxylic acid and fatty acid oxidative capacity, impairs mitochondrial energetics. This study identifies specific contributions of impaired insulin action to mitochondrial dysfunction in the heart.
Key Words: insulin • metabolism • mitochondria • oxidative stress
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Introduction |
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Recent studies have suggested that impaired mitochondrial energetics may contribute to cardiac dysfunction in obesity and diabetes mellitus.1–7 The pathogenesis of mitochondrial dysfunction in obesity or diabetes-related heart disease is likely multifactorial but includes fatty acid (FA)–mediated mitochondrial uncoupling and oxidative damage.3,4,8–11 A commonly associated finding in the heart in experimental models of obesity and diabetes mellitus is myocardial insulin resistance.12–16 However, it is not known whether myocardial insulin resistance per se contributes directly to the pathogenesis of myocardial mitochondrial dysfunction.
Clinical Perspective p 1283
The effects of myocardial insulin signaling on the acute regulation of myocardial metabolism are well known17,18 and include increasing glucose uptake and glycolysis via regulation of GLUT4 translocation19,20 and activation of 6-phosphofructo-1-kinase.21 In perfused hearts, insulin increases glucose oxidation and reduces FA oxidation.13 In vivo, the antilipolytic effect of insulin also reduces the delivery of free FAs to the heart, which further reduces myocardial FA oxidation.18 The direct effects of reduced insulin signaling on myocardial substrate use are not well understood, in part because systemic insulin deficiency or insulin resistance increases the delivery of FA to the heart in vivo, which increases myocardial FA use and activates peroxisome proliferator-activated receptor-
–mediated transcriptional pathways that further augment myocardial FA oxidative capacity.22 In contrast, perinatal loss of insulin receptors in cardiomyocytes reduces myocardial glucose and FA oxidation,23 suggesting that the direct effect of insulin deficiency on myocardial substrate use is distinct from effects that are secondary to loss of insulin signaling in the periphery.
The present study sought to elucidate the direct effects of impaired insulin action on cardiac mitochondria in the absence of systemic metabolic disturbances that accompany insulin resistance or insulin deficiency. Thus, we examined mitochondrial function in mice with perinatal loss of insulin receptors in cardiomyocytes (CIRKO) to test the hypothesis that impaired myocardial insulin action might contribute to mitochondrial dysfunction in the heart in insulin-resistant states. This study demonstrates that genetic deletion of insulin receptors in cardiomyocytes impairs mitochondrial function via multiple mechanisms that include loss of tricarboxylic acid (TCA) cycle and β-oxidation proteins, oxidative stress, and mitochondrial uncoupling. Thus, impaired myocardial insulin signaling might directly contribute to mitochondrial dysfunction in conditions such as obesity and diabetes mellitus.
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Methods |
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Generation of Mice
Mice with cardiomyocyte-selective ablation of the insulin receptor (CIRKO), generated as previously described,23 were fed standard chow (see Materials in the online-only Data Supplement for composition of chow) and housed in temperature-controlled facilities with a 12-hour light/dark cycle (lights on at 6 AM). Animals were studied in the random-fed state during the day (between 7 AM and 1 PM) using protocols approved by the Institutional Animal Care and Use Committee of the University of Utah.
Determination of Cardiac Function by Echocardiography
Echocardiography was performed in lightly anesthetized (isoflurane) mice with a Vivid7 echocardiogram unit (General Electric, Tampa, Fla) by an investigator blinded to genotype and analyzed as previously described.23–25
Cardiac Function MVO2 and Substrate Use in Perfused Mouse Hearts
Retrograde heart perfusions were performed in 8-, 24-, and 54-week-old mice as previously described3 with Krebs buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 MgSO4, 1.2 KH2PO4, 2 CaCl2, and 11 glucose gassed with 95% O2 and 5% CO2. Heart rates were maintained at 360 bpm by pacing at 6 Hz at the level of the atria. Oxygen consumption was calculated as previously described.3 Eight-week-old mouse hearts also were subjected to calcium-induced inotropic stress using our previously described protocols.3 Palmitate oxidation rates and MVO2 were determined in isolated working hearts as previously described.13
Mitochondrial Respiration and ATP Synthesis
Mitochondrial function (oxygen consumption and ATP synthesis rates) were studied in saponin-permeabilized fibers as described3,4,26 and in mitochondria isolated by differential centrifugation27 (see online-only Data Supplement Methods). Substrates used were 5 mmol/L glutamate and 2 mmol/L malate, 10 mmol/L pyruvate and 5 mmol/L malate, or 20 µmol/L palmitoyl-carnitine (PC) with 2 mmol/L malate. To evaluate maximal respiratory capacity of isolated mitochondria, O2 consumption was determined in pyruvate-exposed mitochondria after treatment with oligomycin (1 µg/mL) and the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.7 µmol/L).
Mitochondrial Fractionation
Isolated mitochondria were resuspended in 100 µL of 10 mmol/L Tris/HCl (pH 8.5) and subjected to 3 freeze-thaw cycles with liquid nitrogen. Lysed mitochondria were centrifuged twice at 40 000g for 20 minutes at 4°C. The supernatant (matrix fraction) and the pellet (membrane fraction) were stored/suspended in 10 mmol/L Tris/HCl and maintained at –80°C until used for proteomic analysis by mass spectroscopy or blue-native PAGE (membranes only). The fractionation protocol yields a matrix fraction and a membrane fraction (containing outer and inner mitochondrial membranes) that are enriched for respective representative proteins with minimal cross-contamination (online-only Data Supplement Figure I).
Blue-Native Gel Electrophoresis and In-Gel Complex Activities
Blue-native PAGE was performed as described,28 with some modifications (see online-only Data Supplement Methods).
Mitochondrial Proteomics by Mass Spectrometry
Differentially expressed mitochondrial matrix and membrane proteins were identified with liquid chromatography and mass spectroscopy. Samples were initially subjected to tryptic digestion before liquid chromatography and parallel fragmentation mass spectroscopy (see online-only Data Supplement Methods).
Hydrogen Peroxide Measurement
Mitochondrial hydrogen peroxide (H2O2) generation was measured as previously described4 except that PC 60 µmol/L and L-carnitine 2 mmol/L were used as substrates.
Treatment of Mice With the Antioxidant MnTBAP
Four groups of mice were treated starting at 4 weeks of age. Wild-type and CIRKO mice (n=12 per group) received twice-weekly intraperitoneal injections (20 mg/kg body weight) of the cell-permeable superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride; EMD Chemicals Inc, San Diego, Calif] for 4 weeks. Control wild-type and CIRKO mice (n=12 per group) received twice-weekly intraperitoneal injections of saline. After treatment, animals were euthanized and hearts were collected for determination of aconitase activity and oligomycin-insensitive respiration rates.
Western Blot Analysis
Western blot analysis was performed with total heart or mitochondrial proteins as previously described (see online-only Data Supplement Methods).3
Gene Expression
Total RNA was extracted from 
30 mg heart tissue with Trizol reagent (Invitrogen Corp, Carlsbad, Calif) and purified with the RNEasy Kit (Qiagen Inc, Valencia, Calif). Equal amounts of heart RNA from 6 mice were subjected to real-time polymerase chain reaction with an ABI Prism 7900HT instrument (Applied Biosystems, Foster City, Calif) in 384-well plate format with SYBR Green I chemistry and ROX internal reference (Invitrogen) as previously described.4,25 All reactions were performed in triplicate. Data were normalized to cyclophilin. See online-only Data Supplement Table I for primer sequences.
Electron Microscopy
Ventricular samples were analyzed by electron microscopy, and mitochondrial volume density and number were determined by stereology in a blinded fashion using the point counting method as previously described.4,29 For volume density, 2 images per sample were analyzed (magnification x2000) using 2 grids per image. For mitochondrial number, 3 images per sample were analyzed (magnification x8000).
Statistical Analysis
Data are mean±SEM. Comparisons of a single variable in 
2 age-matched groups were analyzed by 1-way ANOVA followed by Fisher least-protected-squares test (StatView 5.0.1 software, SAS Institute, Cary, NC). In analyses comparing 2 variables such as mitochondrial parameters in control and knockout mice as a function of age, a general linear model (eg, 2-way ANOVA) was used. When significant differences existed across multiple ages, a Tukey-Kramer multiple-comparison adjustment was performed on posthoc comparisons to determine at which ages the measures are different (SAS 9.0.3 software, SAS Institute). For all analyses, values of P<0.05 were considered significant. Unless specified, P values indicate statistically significant difference between groups across all ages. When differences (by posthoc analysis) exist between ages, they are indicated in the figure legends. Proteomic data were analyzed by the use of previously published algorithms.30 In brief, the key algorithm in the Waters Protein Expression System is the clustering algorithm, which chemically clusters peptide components by mass and retention time for all injected samples and performs binary comparisons for each experimental condition to generate an average normalized intensity ratio for all matched accurate-mass, retention-time components. Student t test was used for each binary comparison.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Cardiac Function
Cardiac function was determined by echocardiography and in isolated hearts. We previously reported that fractional shortening and dP/dt (in vivo) were reduced by 20% and 30%, respectively, in 10-week-old CIRKO mice.25 Thus, we determined in vivo cardiac function at later ages. Fractional shortening was reduced by 17% and 7% in CIRKO hearts relative to controls at 27 and 67 weeks of age, respectively (Table 1). In glucose-perfused Langendorff hearts, functional parameters declined with age in CIRKO and control hearts. However, developed pressure, rate-pressure product, and cardiac efficiency were always significantly reduced in CIRKO hearts at the 3 ages examined (8, 24, and 54 weeks; Table 2). Differences in cardiac function between 8-week-old CIRKO and control mice were accentuated under conditions of calcium-induced inotropic stress (online-only Data Supplement Figure II).
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Mitochondrial Dysfunction in CIRKO Mice
Mitochondrial oxygen consumption and ATP production rates in cardiac fibers from 8-, 24-, and 54-week-old CIRKO mice were examined. With glutamate as substrate, maximal ADP-stimulated mitochondrial respirations (VADP) progressively declined with age (Figure 1A). At 8 weeks, CIRKO mice exhibited reduced VADP with pyruvate, which persisted up to 54 weeks and was associated with lower ATP synthesis rates in older animals (24 to 54 weeks; Figure 1B and 1H). With PC, VADP was initially increased in 8-week-old mice but declined progressively with age (Figure 1C). The increased PC respirations at 8 weeks were not associated with increased ATP synthesis rates (Figure 1I). By 24 weeks, ATP synthesis rates declined by 60% and the ATP/O ratio (ATP/VADP) declined by 50% compared with controls (Figure 1I). Thus, mitochondria from CIRKO hearts manifest 2 distinct functional defects: reduced respiratory capacity and ATP generation with all substrates and evidence for mitochondrial uncoupling (decreased ATP/O) when exposed to an FA substrate.
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To confirm the existence of FA-induced mitochondrial uncoupling, studies with pyruvate- or PC-treated isolated mitochondria from 24-week-old mice were performed (Figure 2A through 2D and online-only Data Supplement Figure III). Respiration rates in isolated mitochondria are 10-fold higher than in permeabilized fibers. Under these conditions, the reduction in pyruvate respirations was no longer apparent in CIRKO mitochondria, and no differences were found in oligomycin-insensitive (Voligo) respiration or respiratory control ratios. In contrast, in the presence of the FA substrate PC, Voligo was increased by 1.3-fold, and the respiratory control ratio was proportionately reduced by 33%. Moreover, ATP and ATP/O ratios were reduced, supporting the existence of FA-induced mitochondrial uncoupling.
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To determine whether reduced electron transport chain (ETC) function accounted for mitochondrial respiratory impairment, oligomycin-treated mitochondria were maximally stimulated by exposure to the uncoupling agent FCCP. Under these conditions, oxygen consumption rates are independent of ATP synthesis and reflect maximal ETC capacity. No differences were found between 24-week-old CIRKO and control mitochondria (online-only Data Supplement Figure III). We independently determined in-gel complex I, IV, and V activities after separation of mitochondrial membrane proteins by blue-native gel electrophoresis. No differences in ETC activity were observed at any age (Figure 2E through 2G).
Mitochondrial Proteins, Gene Expression, and Mitochondrial Enzyme Activities
We also tested the hypothesis that defective mitochondrial function may result from changes in the content or activity of enzymes involved in intermediary mitochondrial metabolism that provide reducing equivalents to ETC. First, we conducted a proteomic expression analysis of mitochondrial membrane and matrix compartments (Tables 3 and 4
, online-only Data Supplement Tables IIA and IIB). Ninety-three mitochondrial matrix and 151 mitochondrial membrane proteins were identified. In the mitochondrial matrix, 24 proteins were reduced in CIRKO mice relative to controls. Of these proteins, 10 were involved in FA oxidation, 10 were involved in the TCA cycle, and 3 represented subunits of pyruvate dehydrogenase (PDH) (specifically the E1 subunit and the lipoamide β subunit). In contrast, only 10 largely unrelated proteins were increased in the matrix compartment (Tables 3 and 4, online-only Data Supplement Tables IIA and IIB). In the mitochondrial membrane fraction, FA oxidation proteins also were coordinately reduced, but the pattern for oxidative phosphorylation (OXPHOS) proteins was more variable. For example, protein subunits of complex I, IV, and V were increased, whereas cytochrome C isoforms and subunits of complex III were reduced.
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Second, we examined enzyme activities as a function of age, focusing on the TCA enzyme citrate synthase and the FA oxidation regulatory enzymes carnitine palmitoyl transferase-1 and 3-hydroxyacyl-CoA dehydrogenase. Relative to age-matched controls, citrate synthase activity progressively declined with age in CIRKO mice (online-only Data Supplement Figure IV). Similarly, carnitine palmitoyl transferase-1 and 3-hydroxyacyl-CoA dehydrogenase activities, which were slightly enhanced at 8 weeks, also declined with age in CIRKO mice relative to their age-matched controls (online-only Data Supplement Figure IV).
Third, we conducted a focused analysis of the expression of nuclear-encoded mitochondrial genes and confirmed global reduction in expression of genes involved in cellular and mitochondrial FA uptake and β-oxidation and reduced expression of their transcriptional regulator peroxisome proliferator-activated receptor- (Table 5, online-only Data Supplement Figure V). The mRNA of the E11 subunit of PDH was reduced by 27%. In contrast, genes that regulate mitochondrial biogenesis were essentially unaltered, except for SIRT1 (a potential regulator of PGC-1 activity), the expression of which was reduced by 31%. Consistent with the proteomic analysis, expression of the subunit of complex III (Uqcrc1) was reduced by 20% to 30% (P<0.05), whereas the expression of the complex I subunit (Ndufa9) was not statistically different from controls. Uncoupling protein expression (UCP2 and UCP3) was not increased despite evidence of mitochondrial uncoupling. PDH kinase (PDK4) expression was reduced in 8- and 24-week-old mice, making it unlikely that reduced pyruvate flux resulted from increased phosphorylation of the E1 subunit of the PDH complex. This was confirmed by blotting for phosphorylated PDHE1, which was not altered in CIRKO hearts (online-only Data Supplement Figure VI).
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Contribution of Increased Oxidative Stress to Mitochondrial Phenotypes of CIRKO Mice
To test the hypothesis that oxidative stress contributed to mitochondrial dysfunction and mitochondrial uncoupling, we measured H2O2 generation and the activity of mitochondrial aconitase, the activity of which is susceptible to oxidative stress.31 Mitochondrial aconitase activity was reduced by 38% and 47% in 8- and 24-week-old CIRKO mice, respectively (Figure 3A), in the absence of proportionate differences in aconitase protein levels (data not shown). H2O2 production was increased by 15% and 28% in 8- and 24-week-old CIRKO mice, respectively, compared with age-matched controls (Figure 3B) after exposure to PC. Manganese superoxide dismutase and catalase protein expression was not significantly different between CIRKO and wild-type mice at 8 weeks (data not shown). These data support the hypothesis that increased mitochondrial ROS production could be an early or primary defect in CIRKO mitochondria.
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To test the hypothesis that reactive oxygen species (ROS) may contribute to the increased uncoupling in 8-week-old CIRKO (as evidenced by increased VADP without an accompanying increase in ATP synthesis and increased oligomycin-insensitive respirations [VOligo]), we treated CIRKO mice with the superoxide dismutase mimetic MnTBAP for 4 weeks starting at 4 weeks of age. Treatment with MnTBAP reversed the decline in aconitase activity in both the cytosolic (data not shown) and mitochondrial fractions (Figure 3C) and prevented the increase in VOligo in cardiac fibers from 8-week-old CIRKO mice (Figure 3D). These data suggest that ROS may be partially responsible for mitochondrial uncoupling in CIRKO mouse hearts.
Insulin Signal Transduction
PI3K signaling modulates mitochondrial FA oxidative capacity.32 Thus, we determined whether differences in PI3K signaling pathways contribute to the mitochondrial phenotypes observed. CIRKO mice exhibited a significant increase in the expression of IGF-1 receptors. Moreover, no reduction was found in basal levels of Akt and GSK3β phosphorylation, which trended higher. Whereas insulin stimulation significantly increased Akt and GSK3β phosphorylation in the control hearts, no statistical increase in either Akt or GSK3β phosphorylation after perfusion of CIRKO hearts with 1 nM insulin was observed (online-only Data Supplement Figure VII).
Mitochondrial Number and Ultrastructure Are Altered in CIRKO Mice
Mitochondrial morphology was normal in younger CIRKO mouse hearts, but at 54 weeks, mitochondria appeared dysmorphic with reduced crista density (Figure 4A). Mitochondrial number and volume density were increased at all ages examined (Figure 4B and 4C). These changes occurred despite the absence of any increase in the expression of genes involved in mitochondrial biogenesis and mitochondrial DNA replication such as peroxisome proliferator-activated receptor-
coactivators and β (PGC1, PGC1β) and nuclear respiratory factors (NRF1 or NRF2). Indeed, transcription factor A mitochondrial expression was reduced in 3-week-old mice (Table 5).
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Discussion |
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Perinatal loss of insulin signaling in cardiomyocytes impairs mitochondrial function. An early defect exists in pyruvate use and a progressive reduction in mitochondrial oxidative capacity and ATP synthesis with glutamate and PC substrates. A modest decline in cardiac contractility occurs at an early age, which does not progress to heart failure; however, cardiac function was significantly impaired in vitro after calcium-induced inotropic stress. Two mechanisms for mitochondrial dysfunction were identified. First, a coordinate reduction was found in TCA and FA enzymes that presumably impaired the delivery of reducing equivalents to the ETC, which remained functionally competent. Second, mitochondria from insulin receptor–deficient myocytes were more susceptible to FA-induced oxidative stress and mitochondrial uncoupling. These changes promote a mitochondrial biogenic response that occurs in the absence of increased PGC-1
expression. These data identify an important role for insulin signaling pathways in modulating mitochondrial bioenergetics and integrity. We recently reported that PI3K signaling regulates mitochondrial function in the heart.32 The present study now shows that insulin signaling per se also regulates myocardial mitochondrial oxygen consumption and ATP synthesis rates. The blunted activation of PI3K targets such as Akt and its downstream substrate GSK3β in insulin-perfused CIRKO hearts is consistent with the hypothesis that reduced PI3K signaling could contribute to mitochondrial dysfunction in CIRKO hearts. However, given that basal levels of phosphorylation of these kinases were not reduced, it is also likely that PI3K- or Akt-independent signals downstream of the insulin receptor also play an important role.
The mitochondrial defect appears initially to be specific for pyruvate use and subsequently for FA use. These observations provide a mechanistic basis for our previously published observations that in isolated perfused working hearts from 16- to 20- week-old CIRKO mice, rates of both glucose and FA oxidation were reduced.23 An important mechanism for reduced pyruvate flux appears to be reduced content of 2 key subunits of the PDH complex. The E1 subunit is a key regulator of PDH flux and is the substrate of the regulatory kinase PDK4.33,34 We provide novel evidence that the E11 subunit of PDH may be an insulin-regulated transcript in the heart and that reduced protein levels of this subunit in mitochondrial matrix might occur on the basis of transcriptional repression. Increased phosphorylation of the E1 subunit by PDK4 decreases its stability, whereas blocking degradation of the E1 subunit of PDH increases the activity of and flux through the enzyme complex.35 However, PDHE1 phosphorylation was not increased in CIRKO hearts, and the expression of its kinase (PDK4) was actually reduced. Mitochondrial dysfunction in CIRKO mice is associated with a modest reduction in cardiac function. We previously reported that cardiac function was reduced in isolated working hearts that were perfused with glucose and FA as substrates under normal workload.23 In the present study, we chose to study glucose-perfused hearts because of the defect in pyruvate use in mitochondria. We reasoned that in the presence of glucose alone, contractile dysfunction after calcium-induced inotropic stress would be amplified.
We speculate that an early defect in glucose/pyruvate metabolism could initially lead to the increase in FA use that was observed in the mitochondria of 8-week-old CIRKO mice. However, this cannot be sustained over time because of the coordinate reduction in levels of mitochondrial β-oxidation enzymes. This hypothesis also is supported by the observation of increased rates of FA oxidation in isolated working hearts obtained from 8-week-old CIRKO mice (see online-only Data Supplement Figure VIII) but reduced FA oxidation in 16- to 20-week-old mice.23 The reduction in gene and protein expression levels of a broad array of regulators of FA metabolism was striking and extends our previously reported findings that demonstrated reduced mRNA for acyl CoA dehydrogenases. The likely mechanism for these changes is insulin-mediated regulation of expression levels of the peroxisome proliferator-activated receptor- gene in the heart.
The second major mechanism that contributed to mitochondrial dysfunction is oxidative stress and ROS-mediated mitochondrial dysfunction. Increased ROS production was evident in the hearts of 8-week-old CIRKO mice and was sufficient to reduce the activity levels of the redox-sensitive enzyme aconitase. This increase in ROS also likely reduced mitochondrial energetics by promoting mitochondrial uncoupling as evidenced by reduced ATP/O ratios in PC-treated mitochondria and increased oligomycin-insensitive respiration rates that were normalized by treating animals with the antioxidant MnTBAP. MnTBAP reduces ROS but could increase H2O2 generation, underscoring that mitochondrial superoxide likely mediates the changes observed. Increased ROS could reflect changes in superoxide generation or detoxification. Although increased FA flux could contribute to increased ROS production in 8-week-old CIRKO hearts, it is unlikely to represent the mechanism in older hearts in which FA oxidation is reduced. Proteomic analysis revealed changes in stoichiometry of ETC subunits, which could potentially contribute to increased superoxide generation. A major role for a reduction in ROS degradation pathways as a contributor to increased H2O2 production in CIRKO mitochondria appears unlikely because neither manganese superoxide dismutase levels nor catalase content was changed in 8-week-old mice and catalase content was marginally lower in 24-week-old mice. However, it is possible that reduced TCA flux could limit the supply of reducing equivalents to replenish NADPH pools that maintain antioxidants such as glutathione in the reduced state. Taken together, deficient insulin signaling in the heart likely promotes mitochondrial oxidative stress by multiple mechanisms.
Mitochondrial biogenesis has been described in the hearts of insulin-resistant mice and has been attributed to activation of PGC-1–mediated signaling.36 Here, we show that mitochondrial number and volume density increased in CIRKO mice despite the lack of coordinate changes in mRNA levels of key regulators of the mitochondrial biogenesis pathway such as PGC-1. Thus, the possibility exists that this proliferative response in CIRKO hearts is a consequence of reduced ATP generation or increased oxidative stress, which promote mitochondrial biogenesis. In this regard, it is important to discuss the discrepancy between citrate synthase activities and increased mitochondrial volume density in aging CIRKO mice. Citrate synthase activity is widely used as an indirect estimate of mitochondrial mass. However, in CIRKO mice, our proteomic analyses indicate that citrate synthase protein content in mitochondrial matrix was already significantly lower in 8-week-old CIRKO mice. This observation therefore supports the notion that the morphological "biogenic" response that we observed represents an adaptation to preexisting mitochondrial dysfunction in this model.
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Conclusions |
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We demonstrate that insulin signaling is a regulator of mitochondrial oxidative capacity via mechanisms that may determine TCA cycle flux and the mitochondrial metabolism of pyruvate and FAs. Moreover, impaired insulin signaling predisposes cardiac mitochondria to oxidative stress, which not only might damage mitochondria but also impairs energetics by activating mitochondrial uncoupling. Thus, insulin signaling plays an essential role in the maintenance of mitochondrial homeostasis in the heart. Given the perinatal timing of insulin receptor deletion in CIRKO hearts, it is important to note that metabolic maturation of the heart continues to occur throughout the neonatal period; thus, we cannot rule out that the phenotypes that we have observed might reflect unique effects of insulin resistance during this important developmental window. Future studies in mice with inducible knockout of insulin receptors in adult hearts are required for clarification.
Diabetes mellitus and obesity are independent risk factors for the development of heart failure.37 A growing body of evidence indicates that acquired defects in insulin signaling, which may impair cardiac metabolism and are associated with left ventricular dysfunction, develop in the heart in diabetes mellitus and obesity.38 The present study provides new insights into potential mechanisms linking impaired postnatal insulin signaling with the development of mitochondrial dysfunction in the heart.
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Acknowledgments |
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We thank James Metherall, PhD, for use of robotic facilities for gene expression analysis.
Sources of Funding
This work was supported by grants RO1HL070070, R21DK073590 (funded by the office of Dietary Supplements and the NIDDK), UO1HL70525, and UO1HL087947 from the National Institutes of Health and 19–2006–1071 from the Juvenile Diabetes Research Foundation to Dr Abel, who is an Established Investigator of the American Heart Association, and by a grant from the Department of Veterans Affairs to Dr Litwin. Dr Boudina was supported by postdoctoral fellowships from the Juvenile Diabetes Research Foundation and the American Heart Association. Dr Bugger was supported by a postdoctoral fellowship of the German Research Foundation. Dr Zaha is supported by a postdoctoral fellowship from the AHA; Dr O'Neill, by a physician scientist-training award from the American Diabetes Association; and Dr Palfreyman, by a summer research grant from the Endocrine Society.
Disclosures
None.
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The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.792101/DC1.
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