Phosphofructo-1-Kinase Deficiency Leads to a Severe Cardiac and Hematological Disorder in Addition to Skeletal Muscle Glycogenosis

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm−/−). Here, we show that Pfkm−/− mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm−/− mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm−/− mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm−/− mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies

Bcr/Abl Interferes with the Fanconi Anemia/BRCA Pathway: Implications in the Chromosomal Instability of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34+ cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34+ cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies

Phosphofructo-1-kinase deficiency leads to a severe cardiac and hematological disorder in addition to skeletal muscle glycogenosis

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm−/−). Here, we show that Pfkm−/− mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm−/− mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm−/− mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm−/− mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies

<i>Pfkm^−/−</i> mice develop skeletal muscle glycogenosis and exercise intolerance.

<p>(A) Glycogen storage evidenced by PAS staining in skeletal muscle sections from wild-type (WT) and <i>Pfkm^−/−</i> mice. Scale bar 50 µm. (B) Transmission electron microscopic analysis of skeletal muscle. Arrows show glycogen storage and asterisks point to mitochondria. Scale bar 1 µm. (C) <i>Pfkm^−/− mice</i> showing severe muscle cramps after exercise (5 min). (D) ATP and ADP content was determined in perchloric extracts of skeletal muscle from wild-type (+/+) and and <i>Pfkm^−/−</i> (−/−) mice, in rest and after exercise (5 min), as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. Results are mean±SEM of five mice per group. *<i>P</i><0.05 <i>vs.</i> wild-type.</p

Generation of <i>Pfkm^−/−</i> mice and the effect of <i>Pfkm</i> ablation on skeletal muscle glucose metabolism.

<p>(A) Schematic representation of the wild-type <i>Pfkm</i> locus (top), targeting vector (middle) and targeted allele (bottom). The positions of <i>Hind</i>III (H) and <i>Xba</i>I (X) cleavage sites, the <i>neo</i>^R (neo) and herpes simplex virus thymidine kinase (<i>HSV-tk</i>) genes, and the location of PCR primers used to detect wild-type (PFK-Fw and PFK-Rev) and targeted (Neo and PFK-Rev) alleles are shown. (B) PCR analysis of DNA from wild type (<i>+/+</i>), <i>Pfkm^+/−</i> (<i>+/−</i>) and <i>Pfkm^−/−</i> (−/−) mice using the primers shown in (A). The 0.6 Kb band corresponds to the wild-type allele and the 0.7 Kb band to the mutant allele. (C) Expression of <i>Pfkm</i> in skeletal muscle. Total RNA was obtained from gastrocnemius muscle and analyzed by Northern blot. A representative Northern blot hybridized with a <i>Pfkm</i> probe is shown. (D) PFK activity was determined in skeletal muscle as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. Basal PFK activity in wild-type mice was 36±2.4 U/g tissue. (E–G) Glucose-6-phosphate (E), glucose (F) and glycogen (G) concentrations were determined in perchloric extracts of skeletal muscle from 2–3 month-old wild-type (+/+) and and <i>Pfkm^−/−</i> (−/−) mice, in rest and after exercise (5 min), as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. (H) Serum lactate levels in wild-type (+/+) and <i>Pfkm^−/−</i> (−/−) mice, in rest and after exercise (5 min). Results in D-H are mean±SEM of five to eight mice per group. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> wild-type.</p

Reduction of erythrocyte PFK activity leads to hemolysis, reticulocytosis, and splenomegaly.

<p>(A) PFK activity was determined in blood cell lysates from wild-type (+/+) and <i>Pfkm^−/−</i> (−/−) mice as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. (B,C) Glucose-6-phosphate (B) and 2,3-bisphosphoglycerate (2,3-BPG) (C) concentrations were determined in blood cell perchloric extracts as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. (D–F) <i>Pfkm^−/−</i> show high serum bilirubin levels (D) and reticulocyte number (E,F). New methylene blue stained blood samples were extended on slices (E) and counted (F). Arrows indicate reticulocytes. Scale bar 15 µm. (G,H) Splenomegaly in <i>Pfkm^−/−</i> mice. A high increase in spleen size (G) and weight (H) was observed. Scale bar 5 mm. (I,J) Hematopoietic precursors in cultured cells from spleen (I) and femur (J) from wild-type (+/+) and <i>Pfkm^−/−</i> (−/−) mice. Results are mean±SEM of five to eight mice per group. **<i>P</i><0.01 <i>vs.</i> wild-type.</p

<i>Pfkm^−/−</i> mice show increased skeletal muscle hypoxic markers, vascularization, and fiber necrosis.

<p>(A) The expression of the hypoxia-induced factor (HIF-1α), pyruvate kinase (PK-M), lactate dehydrogenase (LDH), and glucose transporter-1 (GLUT-1) in skeletal muscle of <i>Pfkm^−/−</i> mice was determined by quantitative PCR analysis, as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. Results are mean±SEM of four mice per group. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> wild-type. (B) Skeletal muscle sections showed increased immunostaining for VEGF, leading to hypervascularization, as evidenced by greater immunostaining for endothelial cell marker PECAM-1 (scale bar 25 µm) and collagen IV (scale bar 10 µm). Arrows show blood vessels around muscle fiber. (C) Fiber necrosis in skeletal muscle sections of <i>Pfkm^−/−</i> mice. Arrows indicate cell infiltration of necrotic fibers (scale bar 25 µm). (D) Muscle fiber regeneration is evidenced by multiple centrally located nuclei (arrows) (scale bar 25 µm).</p

Effects of PFKM deficiency in skeletal muscle markers.

<p>(A) Expression of key genes in oxidative metabolism in skeletal muscle of wild-type and <i>Pfkm^−/−</i> mice: Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor δ (PPARδ), carnitine palmytoiltransferase-1 (M-CPT-1), citrate sinthase (CS) and uncoupling protein 2 (UCP-2). (B) Histochemical staining for succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) activities in skeletal muscle of wild-type and <i>Pfkm^−/−</i> mice. Scale bar 25 µm. (C) Expression of myosin heavy chains in skeletal muscle of wild-type and <i>Pfkm^−/−</i> mice: Type I, IIa ,and IIb myosin heavy chains (MyHC-I, MyHC-IIa, MyHC-IIb). (D) Expression of the key genes in skeletal muscle glucose uptake, glucose transporter 4 (GLUT4) and hexokinase-II (HKII), in wild-type and <i>Pfkm^−/−</i> mice. (E) Expression of pentose phosphate pathway genes, transaldolase (TALDO1) and transketolase (TK), in skeletal muscle of wild-type and <i>Pfkm^−/−</i> mice. Relative expression in A, C, D and E was determined by quantitative PCR analysis of total RNA from skeletal muscle, as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000615#s4" target="_blank">Materials and Methods</a>. Results are mean±SEM of five mice per group. *<i>P</i><0.05 <i>vs.</i> wild-type.</p

<i>Pfkm^−/−</i> mice show altered heart glucose metabolism and develop cardiomegaly with age.

<p>(A) PFK activity was determined in heart extracts. Basal PFK activity in wild-type mice was 27.4±5.4 U/g tissue. (B,C) Glucose-6-phosphate (B) and glycogen concentrations (C) were determined in heart perchloric extracts from 2-month-old wild-type (+/+) and <i>Pfkm^−/−</i> (−/−) mice. Results are mean±SEM of five mice per group. *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> wild-type. (D) Transmission electron microscopic analysis of cardiac muscle. Arrows show glycogen storage. Scale bar 2 µm. (E,F) One-year-old <i>Pfkm^−/−</i> mice develop cardiac hypertrophy, evidenced by hematoxilin-eosin staining of heart sections (scale bar 1 mm) (E) and cardiomegaly (F). (G) Longitudinal sections of heart from 3-month-old mice stained with Masson trichromic reagent (scale bar 1 mm). Inset shows septum sections (scale bar 50 µm).</p