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

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

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

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

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.836