Functional deficits in nNOSmu-deficient skeletal muscle: myopathy in nNOS knockout mice

Abstract

Skeletal muscle nNOSmu (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSmu; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSmu. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSmu signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSmu expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.

Figure 1. Sex-specific reductions in body and skeletal muscle mass in nNOS-deficient mice.

The body masses of 8 week old adult male (M) and female (F) mice homozygous for the wild type NOS1 allele (+/+) or NOS1 null allele (−/−) are shown in A. The body masses of wild type males are significantly larger (p<0.01) than KN1 littermates and wild type females (p<0.01) (A). The masses of tibialis anterior (TA) muscles from 8 week old adult wild type males are significantly larger (p<0.01) than KN1 male littermates and wild type females (B). The masses of soleus muscles are similarly affected in nNOS mutant mice where KN1 male solei are significantly smaller (p<0.05) than male littermate controls (C). The soleus muscle of wild type males is larger than that of wild type females (p<0.05). Numbers of animals analyzed: 10–11 wild type males, 11 KN1 males, 10–11 wild type females and 12–13 KN1 females. Values shown are mean±standard error of the mean (S.E.M).

Figure 2. Sex-specific decrease in maximum isometric force-generating capacity in nNOSμ-deficient skeletal muscle.

Maximum isometric force output (P_o) from TA muscles of 8 week old adult male (M) and female (F) mice homozygous for the wild type NOS1 allele (+/+) or or NOS1 null allele (−/−) is shown in A. P_o was significantly decreased (p<0.01) in male KN1 TA muscle compared with sex-matched littermate controls (A). Wild type male mice generated greater total force than wild type female mice (p<0.01). Maximum tetanic force output from female TA skeletal muscle was unaffected by the absence of nNOSμ (A). Specific force (sP_o), maximal tetanic force output normalized for the length and mass of the TA muscle, was not significantly affected by the absence of muscle nNOSμ in both males and females (B). Force-frequency profiles for wild type (black circles) and KN1 mice (grey squares) are shown in C. Males and females are pooled into wild type and KN1 groups. There was no difference in the force-frequency curves between KN1 males and their littermate controls suggesting no gross abnormalities in neuromuscular transmission. Numbers of animals analyzed: 15 wild type (8 males and 7 females) and 15 KN1 (7 males and 8 females). Values shown are mean±standard error of the mean (S.E.M).

Figure 3. nNOSμ-deficient skeletal muscle exhibits decreased resistance to exercise-induced fatigue.

To simulate exercise, TA muscles were maximally stimulated every 2 s for 4 minutes (fatigue period). Force recovery was measured after 1 minute and 5 minutes of rest following the conclusion of the fatigue period. Representative data are shown for wild type (black boxes) and KN1 (grey triangles) and are fitted with exponential decay curves (black for wild type and grey for KN1) (A). Both male and female KN1 mice showed similar patterns of fatigue. During 4 minutes of repeated contraction, nNOSμ-deficient skeletal muscles did not sustain control levels of force (A). Force generation capacity of KN1 mice declined to a significantly lower force plateau (p<0.05) than wild type controls (B). The time taken to fatigue represented by the time constant τ, was not significantly different between wild type and KN1 mice (B). After 1 minute of rest, nNOS-deficient TA muscles did not recover to the same extent as wild type TA muscle (p<0.05). However, after 5 minutes of rest, the force-generating capacity of nNOS-deficient muscles was fully recovered (B). Numbers of animals analyzed: 13 wild type (7 males and 6 females) and 13 KN1 (5 males and 8 females) mice. Values shown in B are mean±standard error of the mean (S.E.M).

Figure 4. nNOSμ-deficiency does not affect the susceptibility of the TA to contraction-induced injury.

Contraction-induced injury was produced by subjecting TA muscles to a series of consecutive lengthening contractions of progressively increasing strain. Strain is the percentage increase in length beyond optimal muscle length Lo. Contractile function is expressed as normalized force (force/initial force or P/Pi). The susceptibility to contraction-induced injury for wild type (black squares) and nNOS-deficient (grey diamonds) TA muscles are shown. There was no significant difference between controls and KN1 littermates. Numbers of animals analyzed: 14 wild type (8 males and 6 females) and 11 KN1 (5 males and 6 females) mice. Values shown are mean±standard error of the mean (S.E.M).