Bé Wieringa

Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity

To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.

Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM–protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (−/−) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre–type dependent atrophy, myotonia, cataract and male–infertility. These results strengthen the contention that simple loss– or gain–of–expression of DMPK is not the only crucial requirement for development of the disease.

Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) -->/<-- ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK-PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast−myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2′-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility

Creatine kinases are important in maintaining cellular‐energy homeostasis, and neuroprotective effects have been attributed to the administration of creatine and creatine‐like compounds. Herein we examine whether ablation of the cytosolic brain‐type creatine kinase (B‐CK) in mice has detrimental effects on brain development, physiological integrity or task performance. Mice deficient in B‐CK (B‐CK–/–) showed no gross abnormalities in brain anatomy or mitochondrial ultrastructure, but had a larger intra‐ and infrapyramidal mossy fibre area. Nuclear magnetic resonance spectroscopy revealed that adenosine triphosphate (ATP) and phosphocreatine (PCr) levels were unaffected, but demonstrated an apparent reduction of the PCr ⇆ ATP phosphorus exchange capacity in these mice. When assessing behavioural characteristics B‐CK–/– animals showed diminished open‐field habituation. In the water maze, adult B‐CK–/– mice were slower to learn, but acquired the spatial task. This task performance deficit persisted in 24‐month‐old, aged B‐CK–/– mice, on top of the age‐related memory decline normally seen in old animals. Finally, a delayed development of pentylenetetrazole‐induced seizures (creating a high‐energy demand) was observed in B‐CK–/– mice. It is suggested that the persistent expression of the mitochondrial isoform ubiquitous mitochondrial CK (UbCKmit) in the creatine/phospho‐creatine shuttle provides compensation for the loss of B‐CK in the brain. Our studies indicate a role for the creatine–phosphocreatine/CK circuit in the formation or maintenance of hippocampal mossy fibre connections, and processes that involve habituation, spatial learning and seizure susceptibility. However, for fuelling of basic physiological activities the role of B‐CK can be compensated for by other systems in the versatile and robust metabolic‐energy network of the brain.

Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice

Apolipoprotein (apo) E is a ligand for the receptor-mediated uptake of lipoprotein remnant particles. Complete absence of apo E in humans leads to a severe form of type III hyperlipoproteinemia. We have used targeted inactivation in murine embryonic stem cells, as also described by others, to specifically study the effects of heterozygous Apoe gene loss on the development of hyperlipidemia. After 6 weeks on a severe semi-synthetic atherogenic diet, heterozygous null mutants, with only one functional Apoe alle, developed hypercholesterolemia as compared with controls (10.1 mM vs. 4.7 mM serum cholesterol). Interestingly, serum cholesterol levels in female heterozygotes were doubled as compared with male heterozygotes (15.0 mM vs. 7.5 mM). On this diet, heterozygous apo E deficient mice also showed an increased susceptibility to atherosclerosis, depending on gender (mean lesion area per section of 9524 microns 2 vs. 61,388 microns 2 for males and females, respectively), whereas wild-type mice displayed far fewer lesions (354 microns 2 and 9196 microns 2 for males and females, respectively). This study indicates that a subnormal expression-level of the Apoe gene leads to hypercholesterolemia and, consequently, to an increased susceptibility to the development of atherosclerosis.

Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

ABSTRACT To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.

Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement

Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1‐catalyzed β‐phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up‐regulation of high‐energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1‐deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1−/− muscles. Thus, AK1‐catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.

TRPM7 Is Required for Breast Tumor Cell Metastasis

TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation.