Cyclophosphamide leads to persistent deficits in physical performance and in vivo mitochondria function in a mouse model of chemotherapy late effects.

Fatigue is the symptom most commonly reported by long-term cancer survivors and is increasingly recognized as related to skeletal muscle dysfunction. Traditional chemotherapeutic agents can cause acute toxicities including cardiac and skeletal myopathies. To investigate the mechanism by which chemotherapy may lead to persistent skeletal muscle dysfunction, mature adult mice were injected with a single cyclophosphamide dose and evaluated for 6 weeks. We found that exposed mice developed a persistent decrease in treadmill running time compared to baseline (25.7±10.6 vs. 49.0±16.8 min, P = 0.0012). Further, 6 weeks after drug exposure, in vivo parameters of mitochondrial function remained below baseline including maximum ATP production (482.1 ± 48.6 vs. 696.2 ± 76.6, P = 0.029) and phosphocreatine to ATP ratio (3.243 ± 0.1 vs. 3.878 ± 0.1, P = 0.004). Immunoblotting of homogenized muscles from treated animals demonstrated a transient increase in HNE adducts 1 week after exposure that resolved by 6 weeks. However, there was no evidence of an oxidative stress response as measured by quantitation of SOD1, SOD2, and catalase protein levels. Examination of mtDNA demonstrated that the mutation frequency remained comparable between control and treated groups. Interestingly, there was evidence of a transient increase in NF-ĸB p65 protein 1 day after drug exposure as compared to saline controls (0.091±0.017 vs. 0.053±0.022, P = 0.033). These data suggest that continued impairment in muscle and mitochondria function in cyclophosphamide-treated animals is not linked to persistent oxidative stress and that alternative mechanisms need to be considered.

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

AIM: Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSµ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. RESULTS: GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSµ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1-/- muscle. Functional analyses of GC1-/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1-/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. INNOVATION: GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. CONCLUSIONS: These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

Volatility of Mutator Phenotypes at Single Cell Resolution.

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

DNA replication error-induced extinction of diploid yeast.

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.

Defects in mitochondrial localization and ATP synthesis in the mdx mouse model of Duchenne muscular dystrophy are not alleviated by PDE5 inhibition.

Given the crucial roles for mitochondria in ATP energy supply, Ca(2+) handling and cell death, mitochondrial dysfunction has long been suspected to be an important pathogenic feature in Duchenne muscular dystrophy (DMD). Despite this foresight, mitochondrial function in dystrophin-deficient muscles has remained poorly defined and unknown in vivo. Here, we used the mdx mouse model of DMD and non-invasive spectroscopy to determine the impact of dystrophin-deficiency on skeletal muscle mitochondrial localization and oxidative phosphorylation function in vivo. Mdx mitochondria exhibited significant uncoupling of oxidative phosphorylation (reduced P/O) and a reduction in maximal ATP synthesis capacity that together decreased intramuscular ATP levels. Uncoupling was not driven by increased UCP3 or ANT1 expression. Dystrophin was required to maintain subsarcolemmal mitochondria (SSM) pool density, implicating it in the spatial control of mitochondrial localization. Given that nitric oxide-cGMP pathways regulate mitochondria and that sildenafil-mediated phosphodiesterase 5 inhibition ameliorates dystrophic pathology, we tested whether sildenafil's benefits result from decreased mitochondrial dysfunction in mdx mice. Unexpectedly, sildenafil treatment did not affect mitochondrial content or oxidative phosphorylation defects in mdx mice. Rather, PDE5 inhibition decreased resting levels of ATP, phosphocreatine and myoglobin, suggesting that sildenafil improves dystrophic pathology through other mechanisms. Overall, these data indicate that dystrophin-deficiency disrupts SSM localization, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity. Together these defects decrease intramuscular ATP and the ability of mdx muscle mitochondria to meet ATP demand. These findings further understanding of how mitochondrial bioenergetic dysfunction contributes to disease pathogenesis in dystrophin-deficient skeletal muscle in vivo.

Reduced coupling of oxidative phosphorylation in vivo precedes electron transport chain defects due to mild oxidative stress in mice.

Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ) treatment of wild type mice) and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1(-/-))) models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O) at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax) was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.