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

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

Volatility of Mutator Phenotypes at Single Cell Resolution

<div><p>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.</p></div

Distribution of single cell mutation counts in mother cells.

<p>(A) Mutation counts of mutator cells during yeast aging. Each lineage is plotted separately, except Lineage G, whose mother cell became multi-budded, producing two distinct lineages: G1 (blue) and G2 (light blue). The gap in Lineage F is due to sequencing failure of one of the daughter clones. <i>Bottom right</i>, all lineages are plotted together, each represented by a different line color. (B) The observed distribution (blue bars, combined data) of mutation counts is plotted against the predicted Poisson distribution based on the average genome-wide mutation rate (2.6 x 10^-7 mutations/bp/cell division) (orange bars, Poisson Model) and a composite distribution resulting from two overlapping Poisson distributions with mutation rates of 4 x 10^-8 (contributing 35%) and 4 x 10^-7 (contributing 65%) mutations/bp/cell division (green bars, Two-Poisson Model).</p

Mutation rate of <i>pol2-4 msh6Δ</i> mother yeast cells at single cell resolution.

<p>(A) Polymerase errors (orange, green, and blue boxes) arising in maternal double-stranded DNA (dsDNA) as mismatches become mutations during S-phase DNA replication (see rectangle) and segregate to the mother (M) or daughter (D) cells. Subscript numbers following M or D indicate the division number that produced the cell (e.g. M₁ is the mother cell after one division). Red arrows indicate only one of several segregation scenarios. Single cell mutation rates (<i>M</i>_<i>1</i><i>μ</i>, <i>M</i>_<i>2</i><i>μ</i>, <i>M</i>_<i>3</i><i>μ</i>) are defined as the number of new mutations fixed in the maternal lineage at each cell division divided by the total number of nucleotides sequenced in all members of a lineage. (B) Genomic distribution of the 237 mutations observed in individual cell divisions (blue lines) among the 16 yeast chromosomes (gray lines). Red lines, centromeres. (C) Mutation spectra of <i>pol2-4 msh6Δ</i> cells from whole genome sequencing (blue) compared to published spectra (red).</p

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

<div><p>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, <i>P</i> = 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, <i>P</i> = 0.029) and phosphocreatine to ATP ratio (3.243 ± 0.1 vs. 3.878 ± 0.1, <i>P</i> = 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, <i>P</i> = 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.</p></div

Co-occurrences of mutations in the same chromosome and cell division.

<p>The random accumulation of 237 mutations over 87 cell divisions was simulated using 10,000 iterations. green histogram, co-occurrences in the simulation with 3 mutations on the same chromosome; green vertical line, actual co-occurrences with 3 mutations; black histogram, co-occurrences with 2 or more mutations on the same chromosome; gray shading, 95% confidence intervals; black vertical line, actual co-occurrences with 2 or more mutations.</p

Skeletal muscle of mice exposed to Cy have evidence of mild oxidative damage at 1 week after exposure.

<p>SDS-PAGE separation of homogenized EDL muscle followed by immunobloting with specific HNE antiserum. Data presented as box plot showing min, median, and max data point. N = 4 for 1 day and 1 week timepoints and N = 8 for 6 week timepoint.</p