Plectin 1d, 1f, 1b, and 1 link desmin IFs with Z-disks, costameres (DGC), mitochondria, and the outer nuclear/ER membrane system, respectively

<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Representative regions of teased EDL fibers from 4-mo-old f-ple and cKO-ple mice stained for proteins as indicated

Arrowheads and arrows indicate Z-disk–aligned and perpendicular longitudinal desmin-positive costameric structures, respectively. In f-ple fibers, note the colocalization of desmin IFs with syncoilin, synemin, cytokeratin 8, β-DG, dystrophin, nNOS, and syntrophin but not with caveolin 3. In cKO-ple fibers, all costameric marker proteins show profoundly changed localization patterns. Bar, 5 μm. (B and C) Quantitative immunoblotting analysis of gastrocnemius lysates from three 6-mo-old mice per genotype (B) and of microsomal fractions from at least three gel runs (C). Loading was normalized to total protein contents (Coomassie-stained gels). Bar graphs represent mean values ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Soleus f-ple (a and c) and cKO-ple (b and d) sections double immunolabeled for plectin and desmin (a and b) or stained for desmin alone (c and d)

Note, desmin aggregates in the fiber interior (d, arrow) and accumulates along the sarcolemma (d, arrowhead) in plectin-negative fibers. The double-headed arrow in panel b represents a plectin-positive fiber with a preserved desmin-positive pattern. (B) f-ple (a, c, and e) and cKO-ple (b, d, and f) heart sections immunolabeled using antibodies to proteins as indicated. In cKO-ple cardiomyocytes, note the aggregates of desmin (b, arrow) and misaligned Z-disks (f, inset) as well as the seemingly preserved intercalated disk structures (double arrows). (C) f-ple (a and c) and cKO-ple (b and d) soleus longitudinal (a and b) and EDL cross sections (c and d) stained for proteins as indicated. Asterisks indicate fibers devoid of IFs in the fiber interior. The double-headed arrow in panel b represents a CNF with preserved IF pattern. The dotted boxes in panels c and d indicate areas shown magnified in the insets. (D) Immunofluorescence microscopy of teased fibers from f-ple (a and c) and cKO-ple (b and d) EDL revealing massive longitudinal desmin aggregates (b) and misaligned α-actinin–positive costameres (d, inset) in cKO-ple mice. No misalignments were observed in the case of f-ple costameres (c, inset). Note also the close association of desmin IFs with f-ple nuclei (a, inset) but their detachment from cKO-ple nuclei (b, inset). Dotted boxes indicate areas shown magnified in insets. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

(A) Longitudinal sections of soleus immunostained using antiserum 46 to plectin

Striated plectin patterns are observed in ple1, ple1b, and dessamples; in ple1d and ple1d/des samples, such patterns are missing. The arrow and arrowheads in the ple1d panel represent plectin-positive sarcolemmal and interior dotlike structures, respectively. Note that the interior of ple1d/des fibers is completely devoid of plectin-positive signals. (B) Teased fibers of EDL were immunostained as in A. Note, the signal associated with longitudinal perinuclear structures was decreased in ple1 compared with ple1b fibers (arrows). Also, costameres were focally disorganized in ple1d and des samples (arrowheads). (C) Ple1d soleus sections double immunolabeled for plectin and desmin (a), desmin and mitochondria (b), or stained for SDH (c). Inset shows subsarcolemmal aggregation of mitochondria in a magnified view of the boxed area. The electron micrograph in panel d shows internal lysis of enlarged mitochondria in the subsarcolemmal region (arrows). (D) Ple1d EDL cross section double immunolabeled for desmin and synemin revealing aggregates in the interior of fibers and largely unaffected sarcolemmal regions (see also inset, a magnified view of the boxed area). (E) Immunofluorescence microscopy of teased ple1d fibers (EDL) using antibodies as indicated. In panels a and b, note the largely unaffected perinuclear and costameric patterns of plectin 1 and 1f, respectively. Panels c and c′ represent sequential confocal sections of one fiber. An optical cross section of this fiber (marked 1) is shown as an inset in panel c′, with horizontal lines indicating the positions of the planes shown in panels c and c′. Note the costameric patterns lacking aggregates in panel c and that desmin aggregates in the interior part of the fiber in panel c′ (arrow). Bars: (A; B; C, a and b; D; and E) 20 μm; (C, c) 50 μm; (C, d) 2 μm. (F) Quantitative immunoblotting of plectin in gastrocnemius lysates from different mouse mutants. Data, relative to WT samples (100%), represent the means ± SEM of three experiments.<p><b>Copyright information:</b></p><p>Taken from "Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms"</p><p></p><p>The Journal of Cell Biology 2008;181(4):667-681.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386106.</p><p></p

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

<div><p>Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.</p></div

UCP2 deficiency does not affect proton leak kinetics or ROS production.

<p>(A) Flow-force relationship in heart mitochondria of 25-week-old wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice examined in the presence of succinate and increasing amounts of malonate (n = 6). Data points indicate mean levels ± standard error of the mean (S.E.M.). (B) Hydrogen peroxide production rate in heart mitochondria in the presence of succinate (S); Pyruvate-Glutamate-Malate (PGM) or Pyruvate-Glutamate-Malate-Succinate (PGMS) as substrates. Measurement in the presence of succinate alone detects reverse flow ROS production from both Complex I and Complex III. When mitochondria are incubated in the presence of pyruvate-glutamate-malate (PGM), the ROS production mainly originates from complex III and therefore it is usually lower. Treatment with Antimycin A (+AA), an inhibitor of CO III, was used as a positive control, to further induce ROS production (n = 4–5). Bars indicate mean level ± standard error of the mean (S.E.M.). (C) Western blot analyses of Manganese Superoxide Dismutase (MnSOD) in heart lysates of 25- and 40-week-old mice (n = 4). (D) Analysis of 4-Hydroxynonenal as measure for oxidative stress induced lipid peroxidation in isolated mitochondria (upper panel) and tissue lysates (lower panel) of 25-week-old mouse hearts. The mitochondrial Complex II 70 kDa protein (C II) was used as loading control in isolated mitochondria and the Heat shock 70 kDa protein 8 (HSC 70) in heart tissue lysates (n = 4). (E) Fold change of <i>Ucp2</i> transcript levels in primary MEFs (P1–P3). (F) ROS production in intact cells was assessed by flow cytometric analyses of primary (passage 1–3) mouse embryonic fibroblasts (MEFs) stained with CM-H₂DCFDA that upon oxidation by ROS, particularly hydrogen peroxide (H₂O₂) and the hydroxyl radical (·OH), yields the fluorescent DCF product. Data are expressed as median values of fluorescence intensity ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

Characterization of mitochondrial cardiomyopathy in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice.

<p>(A) Histological examination of cardiomyocytes from mut and dm mice. TEM – transmission electron micrographs. Arrows indicate lipid droplets, arrowheads abnormal mitochondria. COX/SDH - Enzyme histochemical staining for cytochrome <i>c</i> oxidase (COX) and succinate dehydrogenase (SDH) activities in heart. (B) Quantification of mitochondrial mass in transmission electron micrographs. (C) Relative expression levels of <i>Nppa</i> and <i>Nppb</i>, markers of heart failure. (D) Relative <i>Fgf21</i> mRNA expression levels in heart. (E) Relative <i>Fgf21</i> mRNA expression levels in liver and skeletal muscle. All analyses were performed on 25-week-old mice. Bars indicate mean level ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

UCP2 promotes fatty acid oxidation in mtDNA mutator mitochondria.

<p>(A) Steady-state levels of mitochondrial glucose and fatty acid transporters: insulin-regulated glucose transporter (GLUT4), basal glucose transporter 1 (GLUT1) and mitochondrial carnitine palmitoyltransferase I (CPT1B) in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice. Cytoplasmic calnexin was used as loading control. (B) Quantification of Western blots from (A). (C) Oxygen consumption rates in intact mitochondria in presence of pyruvate-glutamate-malate as substrates. State III (substrates+ADP); State IV (+oligomycin); MAX (+CCCP). (D) Oxygen consumption rates in intact mitochondria in the presence of medium (OC - octanoyl-carnitine) or long chain (PALM - palmitoyl-carnitine) fatty acids as substrates. (E) Maximal oxygen consumption rates upon addition of octanoyl-carnitine+glutamate (OC+Glut) or palmitoyl-carnitine+glutamate (PALM+Glut) to intact mitochondria. (n = 4–6). Bars indicate mean levels ± standard error of the mean (S.E.M.). Statistically significant differences between mut and dm are presented with thick lines. Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

MtDNA mutator mice have lower body mass and upregulated UCP2 levels.

<p>Body weight (A) and daily food consumption (B) in mtDNA mutator (mut) mice in comparison to littermate controls (wt) (n = 11). (C) Northern blot analyses of <i>Ucp2</i> levels in spleen, heart, brain and liver. (D) Fold change of <i>Ucp2</i> transcript levels in spleen, heart, brain, liver and hypothalamus for wt and mut (n = 4). (E) Western blot analyses of UCP2 in spleen and heart mitochondria of wt, mut and UCP2-deficient (ko) mice. (F) Proton motive force (bars) and mitochondrial matrix volume (lines) in liver and spleen mitochondria (n = 3). (G) Mean lifespan of wt, mut, ko and dm mice. All analyses were performed on 25-week-old mice. Bars indicate mean level ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

Characterization of blood metabolites in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice.

<p>(A) Blood glucose and lactate concentrations at 25 weeks of age. (n = 6). (B) Circulating free fatty acid levels in 25- and 40-week-old animals. (n = 5–6). (C) Serum insulin levels of 30-week-old mice. (D) Glucose tolerance test in 30-week-old mice. After 16 hours of starvation period, mice were injected with 2g/kg body weight glucose and clearance was measured after 0, 15, 30, 60 and 120 minutes (n = 7). (E) Insulin tolerance test of 30-week-old random fed mice. Mice were injected with 0.75 U/kg body weight of insulin and glucose levels were measured after 0, 15, 30 60 and 120 minutes (n = 7). (F–L) Analyses of metabolic markers in the serum: (F) Leptin; (G) Ghrelin; (H) Glucagon; (I) Glucagon-like peptide 1 (GLP-1); (J) Glucose-dependent insulinotropic polypeptide (GIP); (K) Plasminogen activator inhibitor-1 (PAI-1); (L) Resistin. All measurements were performed in 30-week-old mice. (n = 4). Bars indicate mean levels ± standard error of the mean (S.E.M.). Statistically significant differences between mut and dm are presented with thick lines. Asterisks indicate level of statistical significance (*p<0.05; **p<0.005; ***p<0.001, Student's <i>t</i>-test).</p