Loss of positive allosteric interactions between neuronal nitric oxide synthase and phosphofructokinase contributes to defects in glycolysis and increased fatigability in muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether the loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that the expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK-specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

Figure 1.

nNOS transgene expression in mdx mouse muscles increases endurance in treadmill running without affecting muscle mass but producing a small shift in fiber-type profile in some muscles. Body mass (A), muscle mass (B and C) and heart mass (D) in 5-month-old mice did not differ in Tg+/mdx and Tg−/mdx mice. Transgene expression also had no effect on the proportion of total fibers that were Type 2 fibers in the gastrocnemius (E) or quadriceps (F) muscles, although there was a small shift from type 2B to type 2A in gastrocnemius (G) but not in quadriceps (H). Although transgene expression did not affect grip strength of the mice (I), expression caused a large increase in endurance in treadmill running time (J). Seven Tg−/mdx mice and 12 Tg+/mdx mice were analyzed for each comparison. * indicates a significant difference at P < 0.001. Error bars, standard error of the mean. Where no error bars are evident, the errors were too small to appear on the scale used for the graph.

Figure 2.

nNOS transgene expression in skeletal muscle does not affect concentration of mitochondrial enzymes or GLUT4, and there is no difference in the hemoglobin content of Tg+/mdx and Tg−/mdx muscles after running. (A) Immunoblots of whole muscle extracts from three, Tg+/mdx mice and three, Tg−/mdx mice were probed with anti-cytochrome C, anti-SDH or anti-GLUT4. The same blots used for incubation with antibodies in (A) were first stained with Ponceau red to show relative loading of each sample (‘Loading’ shown below). (B) Measurements of hemoglobin concentration in muscles of Tg+/mdx and Tg−/mdx mice show that there are no differences in hemoglobin content in the two lines following treadmill running. * indicates significant difference from wild-type controls.

Figure 3.

PFK distribution and activity in muscle. (A) nNOS transgene expression does not affect the concentration of PFK at the cell membrane or in the cytosol of mdx muscle. Immunoblots of soluble and particulate fractions of muscle extracts that were probed with anti-PFK, anti-talin or anti-PIN. The seven left lanes contained the cytosolic fraction, confirmed by the enrichment of PIN and absence of talin in the samples. The seven right lanes contained the particulate fraction enriched in cell membrane, confirmed by the absence of PIN and enrichment with talin in the samples. Lanes 1–4 were samples prepared from Tg−/mdx hindlimb muscles. Lanes 5–7 were prepared from Tg+/mdx muscle extracts. (B) PFK is present in the cytosol of muscle fibers and enriched at the muscle cell membrane. A cross-section of wild-type muscle was immunolabeled with anti-PFK and then second antibody conjugated with a red chromogen (B) and also incubated with FITC-conjugated α-bungarotoxin to label NMJs (arrows) (C). A longitudinal-section of wild-type muscle was immunolabeled with anti-PFK and then second antibody that was conjugated with a red chromogen (D) and an adjacent section similarly treated, except anti-PFK was replaced by PBS only, as a negative control (E). Arrows in Figure 1D indicate the MTJs of two muscle fibers. Bar=50 µm. (F) The specific activity of muscle PFK is positively regulated by nNOS. The loss of nNOS in nNOS null mutants (B) results in a significant reduction in the specific activity of muscle PFK, relative to wild-type muscles (A). Mdx mice (C), which have greatly reduced concentrations of nNOS, also display significant reductions in PFK specific activity in muscle. Expression of a nNOS transgene in wild-type mice (D) or mdx mice (F) causes a significant increase in PFK-specific activity compared with wild-type mice. *significantly differs from wild-type at P < 0.05. (G–I) nNOS, but not eNOS or iNOS, is a positive allosteric regulator of PFK. Activity of PFK was assayed under allosteric conditions in the presence or absence of nNOS (G), eNOS (H) or iNOS (I). Reactions were performed for 2 min at a NOS/PFK molar ratio of 1:1 (solid red line), 1:5 (solid black line), 1:10 (solid blue line), 1:100 (dotted red line) or without NOS (dotted black line). Data shown are representative of three experiments.

Figure 4.

Expression of a nNOS transgene in mdx muscle increases glycogen metabolism during treadmill running. (A) Glycogen content was reduced by nearly 60% in Tg+/mdx mice during treadmill running. In contrast, there was no significant change in glycogen content in Tg−/mdx mice during treadmill running under the same conditions. * indicates significantly different from sedentary mice of same genotype. (B) Lactate content was increased by nearly 50% in Tg+/mdx mice during treadmill running. In contrast, there was no significant change in lactate content in Tg−/mdx mice during treadmill running under the same conditions. * indicates significantly different from sedentary mice of same genotype.

Figure 5.

(A) Graphic representation of the domains of nNOS that used in PFK activity assays. The N-terminal and C-terminal domains and peptides 1, 2 and 3 were cloned and expressed in BL21 (DE3) cells. Peptides 4 through 8 were synthesized commercially. (B) The positive allosteric activity of nNOS lies in the C-terminal domain. Addition of the C-terminal domain of nNOS, but not the N-terminal domain, shows a positive, dose-responsive effect on PFK activity under allosteric conditions. * indicates significantly differs from PFK activity in the absence of nNOS C-terminal or N-terminal domain at P < 0.05.

Figure 6.

The positive allosteric activity of nNOS lies primarily in a 36-amino acid peptide in the FAD-binding domain. Addition of a peptide containing the FAD-binding domain, but not the BH₄/FMN- or NADPH-binding domain, shows a positive, dose-responsive effect on PFK activity under allosteric conditions (A). Peptide 7, a 36-amino acid peptide in the FAD-binding domain of nNOS shows a strong, dose-responsive, positive allosteric effect on PFK activity under allosteric conditions. * indicates significantly differs from PFK activity in the absence of nNOS peptides at P < 0.05.

Figure 7.

Peptide 7 in nNOS lies on the exposed surface of nNOS. The location of the 36-amino acid peptide on nNOS that promotes PFK activity is highlighted in yellow on the image obtained from World Wide Protein Data Bank (http://www.wwpdb.org/) based on the structure determined by Zhang et al. (26).