GM130
nNOS
Overlay
Invitrogen
Sigma-Aldrich
BD Transduction
Labs
Diasorin
Supplemental Figure 1. The Golgi nNOS splice variant is detected by four
distinct pan-specific nNOS antibodies. To confirm that Golgi nNOS labeling was
not due to non-specific antibody cross reactivity we tested the ability of four
commercially available antibodies raised against unique epitopes to detect the
Golgi nNOS. All four antibodies detected the Golgi nNOS. Pan-specific antibodies used were raised against (i) the amino terminus (Invitrogen), (ii) carboxyl
terminus residues 1409-1429 (Sigma-Aldrich), (iii) carboxyl terminus residues
1089-1284 (Becton Dickinson Transduction Labs) and (iv) carboxyl terminus
residues 1414-1428 (Diasorin). FITC-conjugated anti-GM130 antibody was
used to label the Golgi complex.
A
Fiber Number per mm2
400
KN1
Control
p < 0.05
KN2
350
300
250
200
p < 0.05
p < 0.01
p < 0.001
p < 0.001
150
100
50
0
IIa
IIa / IIb
IIb
I / IIa
IIx / IId
Fiber Type
7500
Type IIa Myofiber Area (µm2)
B
Type IIb Myofiber Area (µm2)
I
p < 0.05
p < 0.01
6250
5000
3750
2500
1250
0
WT
KN1
KN2
7500
6250
5000
3750
2500
1250
0
WT
KN1
KN2
Supplemental Figure 2. (A) nNOS splice variants differentially regulate fiber composition in tibialis
anterior (TA) muscle. Antibodies specific for myosin heavy chains 1, IIa and IIb were used to identify slow
oxidative type 1 (most fatigue resistant), fast oxidative type IIa (intermediate fatigue resistance) and fast
glycolytic type IIb (least fatigue resistant) fiber types in the TA muscles from WT, KN1 and KN2 mice.
Unlabeled fibers were designated fast oxidative Type IIx / IId fibers which are also characterized by a
high degree of fatigue resistance. The numbers of fast and slow fibers per mm2 were determined from
entire WT, KN1 and KN2 muscle sections. KN1 muscles exhibited shift to a more fatigue resistant fiber
composition characterized by a reduction in Type II a fibers concomitant with an increase in Type IIx / IId
fibers. In KN2 muscles, there was a 45 % increase in the number of fatigue susceptible fast twitch glycolytic Type IIb fibers. Mean values ± S.E.M are shown. n ≥ 4. (B) The cross sectional areas (CSA) of Type
IIb fibers from KN2 TA muscles were significantly decreased by 31 % relative to KN1 and wild type controls. WT and KN1 Type IIb fiber CSA were not significantly different. Also, the CSA of Type IIa fibers did
not differ significantly between wild type, KN1 and KN2 TA muscles. These data suggest that reduced
muscle mass of KN2 TA muscles may result from inability of Type IIb fibers to attain a normal size. The
median is represented by the horizontal line in the box. n = 4.
C
WT
KN1 KN2 α-SYN
25
B
30
Time to Peak Tension (ms)
p < 0.001
30
Half Relaxation Time (ms)
A
Peak Twitch Force (mN)
p < 0.05
500
450
400
350
300
250
200
150
100
50
0
p < 0.01
p < 0.01
p < 0.01
25
20
15
10
5
0
WT
KN1 KN2 α-SYN
p < 0.05
p < 0.05
20
15
10
5
0
WT
KN1 KN2 α-SYN
Supplemental Figure 3. nNOS splice variants differentially regulate fiber twitch force properties in tibialis anterior (TA) muscle. Because isometric twitch properties depend on muscle
fiber composition, we investigated whether the shifts in fiber composition in KN1 and KN2
mice correlated with changes in the contraction and relaxation times of isometric twitch contractions in TA muscle. (A) Maximum twitch tension (contractility) was decreased significantly in KN2 muscle only. (B) nNOS mutant myofibers contracted at a faster speed,
thereby taking a shorter time to generate peak twitch tension than wild type controls. Faster
times to peak twitch tension are consistent with an increased number of fast twitch fibers (C)
nNOSµ-deficient myofibers exhibited a faster relation time than controls. Mean values ±
S.E.M are shown. n ≥ 6.
A
B
10
9
8
7
6
5
4
3
2
1
0
C
p < 0.01
p < 0.01
p < 0.01
WT
p < 0.01
KN1 KN2 α-SYN
KN1
7500
p < 0.001
6250
p < 0.001
p < 0.001
5000
3750
2500
1250
0
WT
KN1
KN2
KN2
Soleus H & E
WT
Soleus Myofiber Area (µm2)
Soleus mass (mg)
p < 0.001
Supplemental Figure 4. nNOS splice variant regulation of muscle mass is modulated by muscle type.
(A) Mistargeting of sarcolemmal nNOSµ in α-SYN mice or loss of sarcolemmal and cytosolic nNOSµ in
KN1 mice both lead to a significant reduction in soleus muscle mass (contrast with the TA, see Figure 7).
The additional loss of nNOSβ lead to a further significant 50 % reduction (p < 0.01) in muscle mass. (B)
KN1 myofibers were significantly smaller (p < 0.001) in cross sectional area (CSA) than wild type controls
suggesting that nNOSµ could regulate the size of more oxidative muscles. The CSA of KN2 soleus
muscles was significantly smaller than KN1 muscle or wild type controls suggesting that decreased
muscle mass could be accounted for by reduced muscle cell size. (C) Decreased cross sectional area
was evident from hematoxylin & eosin labeled TA muscle sections. Taken together, these data argue that
nNOSβ is a critical regulator of skeletal muscle cell size and nNOSµ regulates the cell size and muscle
mass of more oxidative muscles. (A) Mean values ± SEM for muscle mass. (B) Median is represented by
the horizontal black line in the box. n ≥ 4.
Normalized Force Output (%)
100
90
80
70
60
50
40
30
20
10
0
WT
KN1
KN2
αKO
2
5
10 15 20 25 30 35 40 45
% Stretch Beyond Optimal Length
Supplemental Figure 5. nNOS splice variants do not regulate susceptibility to lengthening
contraction-induced injury. To determine if nNOS was required for protection against
contraction-induced mechanical injury, TA muscles were subject to a series of lengthening
contractions of progressively increasing strain. Strain was induced by stretching TA muscles
from 5 to 45 % beyond their optimal length for generating force (Lo). Increased susceptibility
would be indicated by decreased force output at increasing strains. The susceptibility to
contraction-induced injury in α-SYN (green circles), KN1 (orange squares), and KN2 (red
triangles) did not differ from wild type (blue diamonds). n ≥ 6.
Creatine Kinase Activity U/L
1200
1000
800
600
400
200
0
WT
KN2
Supplemental Figure 6. nNOS splice variant deficiency does not impact myofiber turnover. Skeletal muscle myofiber instability leads to muscle cell turnover which is correlated
with creatine kinase (CK) release into the serum. An unpaired Student’s t-test revealed no
significant difference between mean WT and KN2 serum creatine kinase activity. n ≥ 15.