ABSTRACT
Hypoxia-inducible factor (HIF) transcription factors respond to multiple environmental stressors, including hypoxia and hypoglycemia. We report that mice lacking the HIF family member HIF-2alpha (encoded by Epas1) have a syndrome of multiple-organ pathology, biochemical abnormalities and altered gene expression patterns. Histological and ultrastructural analyses showed retinopathy, hepatic steatosis, cardiac hypertrophy, skeletal myopathy, hypocellular bone marrow, azoospermia and mitochondrial abnormalities in these mice. Serum and urine metabolite studies showed hypoglycemia, lactic acidosis, altered Krebs cycle function and dysregulated fatty acid oxidation. Biochemical assays showed enhanced generation of reactive oxygen species (ROS), whereas molecular analyses indicated reduced expression of genes encoding the primary antioxidant enzymes (AOEs). Transfection analyses showed that HIF-2alpha could efficiently transactivate the promoters of the primary AOEs. Prenatal or postnatal treatment of Epas1-/- mice with a superoxide dismutase (SOD) mimetic reversed several aspects of the null phenotype. We propose a rheostat role for HIF-2alpha that allows for the maintenance of ROS as well as mitochondrial homeostasis.
Subject(s)
Abnormalities, Multiple , Homeostasis/physiology , Neoplasm Proteins , Reactive Oxygen Species , Trans-Activators/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Hypoxia , Electron Transport Complex IV , Gene Expression Regulation , Heart/physiology , Homozygote , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Mimicry , Muscle, Skeletal/ultrastructure , Oxidative Stress , Peroxidases , Peroxiredoxin III , Peroxiredoxins , Superoxide Dismutase , Superoxides , Survival Rate , Trans-Activators/deficiency , Trans-Activators/genetics , TransfectionABSTRACT
Tissue engineering represents a potential method for repairing damaged skeletal muscle tissue. Extracellular matrix (ECM) proteins were evaluated for their ability to aid in cell attachment, whereas a poly(L-lactic acid) (PLLA) fiber scaffold was tested as a substrate for the differentiation of human skeletal muscle cells. In comparison to uncoated or gelatin-coated PLLA films, cell attachment increased significantly (p < 0.001) on PLLA films coated with ECM gel, fibronectin, or laminin. Myoblasts differentiated into multinucleated myofibers on ECM gel-coated PLLA fibers, and expressed muscle markers such as myosin and alpha-actinin. Oligonucleotide microarray analysis showed similar gene expression profiles for human skeletal muscle cells on ECM gel-coated PLLA fibers as to that observed for myofibers on tissue culture plates. Therefore, PLLA fibers coated with ECM proteins provide a scaffold for the development of skeletal muscle tissue for tissue engineering and cell transplantation applications.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation/physiology , Extracellular Matrix Proteins/metabolism , Lactic Acid/metabolism , Muscle, Skeletal/cytology , Myoblasts, Skeletal , Polymers/metabolism , Animals , Cell Adhesion/physiology , Cell Line , Gene Expression Profiling , Gene Expression Regulation , Humans , Lactic Acid/chemistry , Materials Testing , Mice , Muscle, Skeletal/physiology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/physiology , Oligonucleotide Array Sequence Analysis , Polyesters , Polymers/chemistry , Surface Properties , Tissue Engineering/methodsABSTRACT
Endurance exercise training promotes mitochondrial biogenesis in skeletal muscle and enhances muscle oxidative capacity, but the signaling mechanisms involved are poorly understood. To investigate this adaptive process, we generated transgenic mice that selectively express in skeletal muscle a constitutively active form of calcium/calmodulin-dependent protein kinase IV (CaMKIV*). Skeletal muscles from these mice showed augmented mitochondrial DNA replication and mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism and electron transport, and reduced susceptibility to fatigue during repetitive contractions. CaMK induced expression of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a master regulator of mitochondrial biogenesis in vivo, and activated the PGC-1 gene promoter in cultured myocytes. Thus, a calcium-regulated signaling pathway controls mitochondrial biogenesis in mammalian cells.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 4 , Calcium-Calmodulin-Dependent Protein Kinases/genetics , DNA Replication , DNA, Mitochondrial/biosynthesis , Electron Transport , Fatty Acids/metabolism , Gene Expression , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria, Muscle/enzymology , Muscle Contraction , Muscle Fatigue , Muscle Fibers, Skeletal/ultrastructure , Muscle, Skeletal/enzymology , Muscle, Skeletal/ultrastructure , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Transgenes , Up-RegulationABSTRACT
Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection. Among 16,267 gene elements surveyed, 3,532 elements showed at least a 2.5-fold change at one or more time points during a 14-day time course. Hierarchical cluster analysis and semiquantitative reverse transcription-PCR showed induction of genes important for cell cycle control and DNA replication during the early phase of muscle regeneration. Subsequently, genes for myogenic regulatory factors, a group of imprinted genes and genes with functions to inhibit cell cycle progression and promote myogenic differentiation, were induced when myogenic stem cells started to differentiate. Induction of a majority of these genes, including E2f1 and E2f2, was abolished in muscles lacking satellite cell activity after gamma radiation. Regeneration was severely compromised in E2f1 null mice but not affected in E2f2 null mice. This study identifies novel genes potentially important for muscle regeneration and reveals highly coordinated myogenic cell proliferation and differentiation programs in adult skeletal muscle regeneration in vivo.