Decreases in Specific Force and Power Production of Muscle Fibers from Myostatin-Deficient Mice are Associated
with a Suppression of Protein Degradation
+1,2,3Mendias C L; 3Kayupov E; 3Bradley J R; 3,4Brooks S V; 5Claflin D R
+1School of Kinesiology and Departments of 2Orthopaedic Surgery, 3Biomedical Engineering, 4Molecular & Integrative Physiology and 5Plastic Surgery
University of Michigan, Ann Arbor, MI
cmendias@umich.edu
INTRODUCTION
Myostatin is a member of the TGF-β superfamily of cytokines and is
a negative regulator of skeletal muscle mass. Compared with wild type
mice (MSTN+/+), mice with an inactivation of myostatin (MSTN-/-) have
an up to two-fold increase in muscle mass. Studies of C2C12 myotubes
indicate that inhibiting myostatin might increase muscle mass by
decreasing the expression of the E3 ubiquitin ligase atrogin-1, which is
an important rate limiting enzyme in muscle protein degradation. At the
whole muscle level, EDL muscles of MSTN-/- mice have greater
maximum isometric force production (Fo), but have a decrease in
specific maximum isometric force (sFo, Fo normalized to muscle crosssectional area). To gain a greater understanding of the influence of
myostatin on muscle contractility, we determined the impact of
myostatin deficiency on (i) the contractile properties of permeabilized
single muscle fibers and (ii) the expression of atrogin-1 and the content
of ubiquitin-tagged myosin heavy chain in whole muscle tissue. We
hypothesized that, compared with MSTN+/+ mice, single fibers from
MSTN-/- mice would have a greater Fo, but no difference in sFo or power
production, and that MSTN-/- mice would have a decrease in ubiquitintagged myosin heavy chain and atrogin-1 gene expression.
METHODS
All experiments were conducted with IACUC approval. The strain of
myostatin-deficient mice used in this study was originally generated by
Dr. Se-Jin Lee. Male mice aged 5 - 6 months were used in this study.
Single Fiber Contractility. Contractility measurements were made
using permeabilized single fiber segments obtained from EDL muscles.
Each fiber was attached at one end to a force transducer and at the other
end to a servomotor and adjusted to a fiber length (Lf) corresponding to
an average sarcomere length of 2.5µm. Fibers were exposed to a high[Ca2+] activating solution to elicit Fo. While activated, a 30% strain was
applied to induce damage, evaluated as the reduction in force (%Fo)
observed following the injury-inducing contraction. The fiber CSAs
were used to calculate sFo (sFo = Fo × CSA-1). Force-velocity
characteristics were evaluated by applying a series of constant-velocity
shortening movements to the activated fiber. Following each shortening
movement, the fiber was returned to Lf and the next shortening
movement was applied. The force during each constant-velocity
shortening period was measured and a rectangular hyperbola was fitted
to the resulting velocity-force data. The intersection of the fitted curve
with the velocity axis was defined as Vmax (Lf × s-1) and the velocity at
which the curve passed through a force equivalent to 2.5% of Fo was
defined as V2.5 (Lf × s-1). Maximum power generating capacity was
calculated from the parameters of the fitted curve and then divided by
fiber volume to obtain Pmax (W × l-1).
SDS-PAGE and Immunoblot. EDL muscles were homogenized in
sample buffer and equal amounts of protein were loaded into mini-gels
and subjected to electrophoresis. Coomassie Brilliant Blue was used to
detect total myosin heavy chain protein. To detect ubiquitinated myosin
heavy chain, gels were blotted and probed with an HRPO tagged antiubiquitin antibody.
Gene Expression. RNA was isolated from EDL muscles, treated with
DNase I and reverse transcribed. cDNA was amplified using primers for
atrogin-1 and β2-microglobulin (β2m) in a real-time thermal cycler.
Statistical Analysis. Results are presented as mean ± SE.
Differences were tested with Student's t-test with α = 0.05.
RESULTS
CSA (µm2)
Fo (mN)
sFo (kPa)
Injury Force
Deficit (% Fo)
Vmax (Lf × s-1)
V2.5 (Lf × s-1)
Pmax (W × l-1)
MSTN+/+
2520±140 (37)
0.207±0.016 (26)
86.2±4.4 (26)
8.07±1.99 (10)
MSTN-/3040±170* (36)
0.201±0.015 (25)
70.7±4.7* (25)
8.94±2.36 (10)
3.04±0.18 (11)
2.48±0.12 (11)
16.7±1.2 (11)
3.46±0.22 (11)
2.64±0.13 (11)
11.8±1.4* (11)
Table 1. Muscle
fiber contractility
values.
Mean±SE (n).
* significantly
different from
MSTN+/+
(p < 0.05).
Figure 1. Force-Velocity (A) and Power-Velocity (B) curves. N = 11
fibers for each genotype. Each point represents mean ± SE.
Figure 2. Deficiency in protein
degradation in EDL muscles of
MSTN-/- mice. Compared with
MSTN+/+ mice, there is a
decreased amount of ubiquitintagged myosin heavy chain (A)
and a decrease in atrogin-1
expression (B) in EDL muscles
from MSTN-/- mice. N = 4 mice
per genotype, * significantly
different from MSTN+/+ (p <
0.05).
DISCUSSION
The results of this study provide new insight into the role of
myostatin in modulating skeletal muscle contractility. In agreement
with prior data obtained from histological studies, the CSA of muscle
fibers from MSTN-/- mice was greater than MSTN+/+ mice. Despite
having larger muscle fibers, there was no difference in Fo values
between the two genotypes and both exhibited similar force-velocity
characteristics. Consequently, MSTN-/- mice had a lower sFo and
normalized power output than MSTN+/+ mice. The MSTN-/- mice also
had a decrease in atrogin-1 expression and ubiquitinated myosin heavy
chain. Taken together, these findings suggest that the increase in muscle
mass that results from the inhibition myostatin occurs, at least in part, by
decreasing protein degradation. The ubiquitin-proteasome system is the
major pathway for the breakdown and recovery of damaged and
misfolded proteins in muscle fibers. The increase in muscle fiber size
with accompanying decrease in sFo and power in MSTN-/- mice are
consistent with an accumulation of damaged or misfolded proteins that
have yet to be hydrolyzed.
Because myostatin inhibition can result in a substantial increase in
total muscle mass, there has been much interest in the development of
therapeutic inhibitors of myostatin for the treatment of a wide variety of
muscle wasting diseases. While the results of the current study indicate
that myostatin inhibition in healthy mice causes an increase muscle fiber
size without increasing force production, this does not necessarily mean
that therapeutic inhibition of myostatin cannot be beneficial in treating
muscle wasting diseases. For diseases that involve an upregulation of
atrogin-1, myostatin inhibition may help to reduce muscle atrophy and
decrease strength loss. The inhibition of myostatin for ergogenic
purposes, however, is not supported.
ACKNOWLEDGEMENTS
This study was supported by grants from the NSF AGEP Program
(0450063) and NIAMS (AR055624).
Paper No. 45 • 56th Annual Meeting of the Orthopaedic Research Society