Download ASCB2004 - Clinical Neurophysiology

Down-regulation of Actin and Myosin mRNA in Animal Models of Acute
Quadriplegic Myopathy
of Clinical
Neurophysiology,
Uppsala University,
Sweden
2Center for
Developmental and
Health Genetics ,
Pennsylvania State
University,
University Park, PA,
USA
3Hershey Medical
Center,
Pennsylvania State
University, Hershey,
PA, USA 4Dept. of
Anesthesiology,
Karolinska Institute
and Linköping
University, Sweden
The study was supported
by NIH (AR45627,
AR47318, AG14731) and
the Swedish Research
Council (8651)
EDL
Soleus
140
140
120
120
100
100
80
80
%
%
•Compared with controls, there was a decrease in
mRNA expression of skeletal muscle proteins, actin and
myosin, in both the rat and pig model of AQM
• In the rat model, a decrease in myosin binding protein
C and an increase in myosin binding protein H mRNA
expression was found
•A decrease in muscle weight and myosin:actin ratio was
found in the rat model
60
60
40
40
20
20
0
0
MyHCIIx
MyHCIIb
MyHCIIa
140
140
120
120
100
100
80
%
80
INTRODUCTION
Acute quadriplegic myopathy (AQM):
•secondary disease seen in intensive care unit patients on
the mechanical ventilator with sepsis and receiving
neuromuscular blocking agents (NMBA) and/or
corticosteroids (CS)
•characterized by intact cognitive and sensory
function, symmetric paralysis/weakness of spinal
cord innervated muscles, decreased muscle
membrane excitability and decreased amplitude of
CMAP
• major observations in patients include a general
decrease in myofibrillar content, a specific but
variable loss of myosin and myosin associated
proteins, very low thick-filament to thin-filament
ratios, an absence of myosin mRNA and a
dramatically impaired force-generating capacity in
AQM muscle cells (Larsson et al, 2000)
To investigate the mechanisms underlying AQM we
developed two experimental mammalian models, i.e.
porcine and rat model.
METHODS
• Animals were mechanically ventilated and exposed to
various combinations of CS, NMBA and sepsis
• Quantification of the expression of skeletal muscle
proteins actin and myosin mRNA using RT-PCR
• Quantification of myofibrillar proteins using SDS-PAGE
and confocal microscopy
• Fiber size analysis using enzyme histochemistry
FIGURE 1
A.
60
60
NMBA, CS, and
Sepsis
B.
CS Only
MyHCTypeI
Masseter
Diaphragm
40
40
20
20
0
0
MyHCIIx
MyHCIIa
MyHCIIb
MyHCTypeI
MyHCIIx
120
Control
Denervated
Mechanically Ventilated
100
80
60
Myosin mRNA expression in the EDL, soleus, diaphragm and masseter
muscles of control, denervated and neuromuscular blocked
mechanically ventilated rats.
40
20
0
1
Day 1
Day 5
Day 1
Day 5
3
5
Day
Control
CS
NMBA, CS and Sepsis
FIGURE 4
Myosin : Actin Ratio by SDS-PAGE
3.0
A) SDS-PAGE (12%) of pig gluteus maximus muscle in NMBA, CS and Sepsis treated animal and
an CS only exposed animal. B) Myosin mRNA expression on days 1, 3, and 5 for pigs treated
with varying combinations of NMBA, CS and/or Sepsis.
Control
Denervated
Mechanically Ventilated
2.5
2.0
FIGURE 2
A
Ratio
1Dept.
Acute quadriplegic myopathy (AQM), which is characterized by symmetrical weakness and/or paralysis of spinal
nerve innervated muscles and intact sensory and cognitive function, affects a large percentage of patients requiring
mechanical ventilation. However, the mechanisms underlying AQM are poorly understood, but clinical studies have
shown a complete or partial loss of the motor protein myosin and a block of myosin synthesis at the
transcriptional level. The diagnosis of AQM is typically made after the disease has progressed to the later stages,
which makes understanding the cellular and molecular mechanisms of the disease difficult. To better understand
the initial mechanisms of AQM, two experimental animal models have been developed in the rat and pig. Specific
interest is focused on the expression and content of the myofibrillar proteins actin and myosin using confocal
microscopy and SDS-PAGE gel electrophoresis and mRNA expression using real-time PCR. In accordance with
previous findings from patients with AQM, we observe a decrease in myofibrillar protein mRNA expression in
spinal nerve innervated muscles such as the extensor digitorum longus, soleus and biceps femoris. Cranial
innervated muscles (e.g., masseter) and those muscles passively activated (e.g., diaphragm) were less affected.
Additionally, there was a decrease in myofibrillar protein expression as seen using SDS-PAGE gel electrophoresis.
In conclusion, this suggests that the rodent and porcine experimental models will be very valuable in future
mechanistic studies on the molecular mechanisms underlying the development of AQM. Furthermore, our data
support the idea that the down-regulation of myosin and actin mRNA may play a role in the underlying
mechanisms of AQM.
%
H.
J. Nordquist1,
P. Andersson1,
K. Kandala2,
H. Zackrisson4,
L. Eriksson4,
S. Walther4,
B. Dworkin3,
L. Larsson1,2
ABSTRACT
%
Norman1,
FIGURE 3
RESULTS
a
b
B
c
1.5
1.0
0.5
d
e
f
0.0
EDL
Soleus
Diaphragm
Masseter
Muscle
g
h
Myosin : Actin ratio on 12% SDS-PAGE for the EDL, soleus, Diaphragm and
masseter of control, denervated and neuromuscular blocked mechanically
ventilated rats.
i
(A) Myofibrillar ATPase (a-c), hematoxylin-eosin (d-f), and NADH (g-h)
stainings of control, CS only and NMBA, CS and sepsis exposed pigs. (B)
Confocal image of a single fiber stained for myosin.
TABLE 1
Soleus
EDL
Tibialis
Anterior
NMB
76 ± 17**
97 ± 21*
369 ± 66*
Control
134 ± 22
129 ± 17
569 ± 22
Muscle weights (in grams) for the soleus, EDL and tibialis anterior muscles of
neuromuscular blocked mechanically ventilated and control rats.
CONCLUSION
•The rodent and porcine experimental AQM models offer an
excellent opportunity to unravel the mechanisms underlying
AQM in ICU patients, i.e., the loss of myofibrillar proteins
and the impaired synthesis of myosin at the transcriptional
level confirm previous clinical observations.
•These two experimental models will be used extensively in
parallel with clinical studies on patients with AQM. Specific
interest is focused on (1) the link between membrane
depolarization and transcription, (2) quantitative protein and
mRNA analyses in response to different interventions, such
as sepsis, NMBA and CS