Biosci. Rep. (2010) / 30 / 41–50 (Printed in Great Britain) / doi 10.1042/BSR20080150
Low dose simvastatin induces compositional,
structural and dynamic changes in rat skeletal
extensor digitorum longus muscle tissue
Nihal SIMSEK OZEK*, Yildirim SARA†, Rustu ONUR† and Feride SEVERCAN*1
*Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey, and †Department of Pharmacology, Faculty of
Medicine, Hacettepe University, Ankara, Turkey
'
$
Synopsis
Statins are commonly used drugs in the treatment of hypercholesterolaemia. There are many adverse effects
of statins on skeletal muscle, but the underlying mechanisms remain unclear. In the present study, the effects of low
dose (20 mg/kg) simvastatin, a lipophilic statin, on rat EDL muscle (extensor digitorum longus muscle) were investigated at the molecular level using FTIR (Fourier-transform infrared) spectroscopy. FTIR spectroscopy allows us rapid
and sensitive determination of functional groups belonging to proteins, lipids, carbohydrates and nucleic acids simultaneously. The results revealed that simvastatin treatment induces a significant decrease in lipid, nucleic acid, protein
and glycogen content. A significant increase in the lipid/protein and nucleic acid/protein ratios was also obtained
with simvastatin treatment. Furthermore, an increase in lipid order and membrane fluidity was detected. A decrease
in the bandwidth of the amide I band and shifting of the position of this band to higher frequency values in treated
muscle indicates structural changes in proteins. Detailed secondary structure analysis of the amide I band revealed
a significant increase in antiparallel and aggregated β-sheet, random coil structure and a significant decrease in
β-sheet structure, which indicates protein denaturation.
Key words: extensor digitorum longus muscle (EDL muscle), Fourier-transform infrared (FTIR) spectroscopy, lipid
order, membrane fluidity, statin, simvastatin
&
INTRODUCTION
Statins, also known as HMG-CoA (hydroxy-3-methylglutarylCoA) reductase inhibitors, are the most commonly used lipidlowering drugs. They block cholesterol biosynthesis by inhibiting HMG-CoA reductase enzyme. They are widely used in the
treatment of hypercholesterolaemia [1–3]. Even though statins
are clinically in common use, some of their adverse effects were
reported in skeletal muscle. These myotoxic effects may range
from myalgia and myopathy to potentially life-threatening rhabdomyolysis [1]. The incidence of these myotoxicities is known to
be dose dependent. In a previous study, the incidence of myopathy
with patients receiving 20 mg of simvastatin/day was found to be
0.02 %, whereas this rate was observed to be 0.4 % in patients
receiving 40 or 80 mg of simvastatin/day [4]. Moreover, the prevalence of these symptoms increases when statins are used with
other drugs such as cyclosporin, fibrate and gemfibrozil. Additionally, the lipophilic or hydrophilic properties of statins also
affect the incidence of these adverse effects in skeletal muscle
%
since the lipophilic statins enter the cell by simple diffusion especially in non-hepatic tissues [5].
Little is known about the mechanisms of statin-induced
skeletal-muscle injury. Statins lead to apoptosis by reducing the
amount of small GTP-binding proteins [2]. They disrupt cellular
respiration and energy metabolism by decreasing synthesis of
ubiquinone (coenzyme Q10), an essential element of mitochondria [6]. It was also reported that statins affect membrane fluidity
and its electrical properties by reducing the cholesterol content
of skeletal-muscle membranes [7].
Simvastatin is more myotoxic than pravastatin due to its
lipophilicity resulting in its greater uptake by myocytes [5].
Moreover, simvastatin has the second highest incidence rate in
rhabdomyolysis, which is one of the myotoxic effects of statins
[8]. The number of biochemical and morphological studies on
simvastatin–skeletal muscle interactions is very limited. There
is agreement in the literature that the protein and nucleic acid
content are decreased as a result of simvastatin treatment [5,9].
However, there are conflicting results about the drug-induced
changes in the amount of lipids [10,11]. Despite these studies
.................................................................. ............................................................. ................................................................. .............................................................. ..............................................
Abbreviations used: FTIR, Fourier-transform infrared; EDL muscle, extensor digitorum longus muscle; HMG-CoA, hydroxy-3-methylglutaryl-CoA.
1 To whom any correspondence should be addressed (email [email protected]).
www.bioscirep.org / Volume 30 (1) / Pages 41–50
41
N. Simsek Ozek and others
the effects of this drug on the structure and function of macromolecules in rat EDL muscle (extensor digitorum longus muscle)
have not been reported before and these effects need to be clarified at the molecular level. Therefore, in the present study,
we studied the effect of simvastatin on rat EDL muscle to better understand its molecular mechanism. To achieve this, we
performed FTIR (Fourier-transform infrared) spectroscopy and
determined simvastatin-induced alterations in the concentration,
content, structure and dynamics of macromolecules, which, to
the best of our knowledge, have not been reported previously.
FTIR spectroscopy is a non-destructive, rapid and sensitive technique that simultaneously monitors different functional
groups belonging to macromolecules of biological tissues. Using this technique we can detect even very small variations and
early alterations in disease conditions [12]. The shifts in peak
positions, changes in bandwidth and area values of the bands
give valuable structural and functional information on biological
materials [12–16].
Model 77520) to remove the free water. The dried samples were
ground with liquid nitrogen in a liquid nitrogen-cooled colloid
mill (Retsch MM200) to obtain tissue powder. The tissue powder
was mixed with dried potassium bromide (KBr) in a mortar (in
the ratio 0.5:150) and dried further in the freeze-drier for 18 h to
remove all traces of the remaining free or unbound water. The
mixture was compressed into a thin KBr disc under a pressure
of ∼100 kg/cm2 producing transparent discs for FTIR studies
[12,13].
The measurement of serum cholesterol levels was performed
by HPLC as reported in detail by Yilmaz et al. [17].
FTIR spectra and data analysis
Adult male Wistar rats (12–14 weeks) weighing 250–300 g
(Experimental Animal Center, Hacettepe University, Ankara,
Turkey) were selected randomly. The rats were fed with a standard
diet with water ad libitum, and kept in a conventional room with
◦
controlled light (12 h light/12 h dark), temperature (22 +
− 1 C),
relative humidity (40–50 %) and ventilation (15 air changes per
hour). They were allowed to adapt to their environment for 1 week
prior to the experiments. All procedures used in the experiments
were approved by the Ethics Committee of Hacettepe University.
The rats were separated into two groups as control (n = 10)
and simvastatin-treated (n = 8). The control group received serum
physiological solution. Simvastatin in serum physiological solution (20 mg/kg per day) was given to the simvastatin treatment
group daily by intragastric intubations using stainless curved
feeding needle (18 ga, 3 in; Stoelting) for 30 days and they
were weighed weekly. At the end of the treatment period, rats
were anaesthetized with diethyl ether. Blood samples were drawn
to measure serum cholesterol levels by using HPLC, and EDL
samples were then dissected and stored at –80◦ C for experimental
analysis.
IR spectra of EDL samples were obtained using a PerkinElmer
Spectrum 100 FTIR spectrometer (PerkinElmer, Norwalk, CT,
U.S.A.) equipped with a MIR TGS detector. The interfering
spectrum of air and KBr transparent disc was recorded as background and subtracted automatically by using appropriate software (Spectrum 100 software). The spectra were recorded in the
4000–400 cm−1 region at room temperature (21◦ C). A total of 50
scans were taken for each interferogram at 4 cm−1 resolution.
Collections of spectra and data manipulations were carried out
using Spectrum 100 software (PerkinElmer). Each sample was
scanned as three different replicates under the same conditions,
all of which gave identical spectra. The average spectra of these
three replicates were then used in detailed data and statistical analysis. Using the same software, the spectra were first smoothed
with nine-point Savitsky–Golay smooth function to remove the
noise. The average spectra were baseline-corrected and normalized with respect to specific bands for visual demonstration. During the determination of the mean values of the peak positions,
band area and bandwidth, the average spectra belonging to the
individuals of each group, were considered. The band positions
were measured using the frequency corresponding to the centre
of weight from raw spectra. The band areas were calculated from
smoothed and baseline-corrected spectra using Spectrum software. The bandwidth value of amide I band was calculated as the
width at 0.75× height of the absorption signal in terms of cm−1
from raw spectra. Moreover, the bandwidth of the CH2 asymmetric stretching band was calculated from 0.75× height of both
absorption and well-resolved second derivative spectra in terms
of cm−1 .
For the determination of simvastatin-induced protein secondary structure variations, amide I band was used and detailed
analysis was carried out using the OPUSNT data collection software package (Bruker Optics, Reinstetten, Germany). Firstly, the
second derivatives spectra were obtained by applying a Savitzky–
Golay algorithm with nine smoothing points and these derivatives
vector normalized at 1700–1600 cm−1 and then the peak intensities were calculated. The peak minima of the second derivative
signals were used, since they correspond to the peak positions of
the original absorption spectra [12].
Sample preparation for FTIR and HPLC studies
Statistics
The EDL samples were dried overnight in a Labconco freezedrier (Labconco FreeZone® , 6 litre Benchtop Freeze Dry System
The results were expressed as means +
− S.D. The differences
in means were analysed statistically using a non-parametric
MATERIALS AND METHODS
Materials
Simvastatin (Zocor) was purchased from Merck, Sharp
and Dohme (West Point, PA, U.S.A.) as tablets, each of
40 mg. Potassium bromide (KBr) was obtained from Merck
(Darmstadt, Germany). All chemicals were obtained from commercial sources at the highest grade of purity available.
Preparation of experimental animals
..........................................................................................................................................................................................................................................................................................................................................................................
42
C The
Authors Journal compilation
C 2010
Biochemical Society
Effects of low dose simvastatin on rat skeletal EDL muscle tissue
Table 1 General band assignment of the FTIR spectrum of skeletal-muscle tissue based on the literature [12–16,38,45]
Peak no.
Wavenumber (cm−1 ) Definition of the spectral assignment
1
3450
Intramolecular H-bonding
2
3330
Mainly N-H stretching (amide A) of proteins with a little contribution from O-H stretching of polysaccharides
and intermolecular H-bonding
3
3062
C-N and N-H stretching (amide B) of protein
4
3014
Olefinic =CH stretching vibration: unsaturated lipids, cholesterol esters
5
2962
CH3 asymmetric stretching: mainly lipids, with a little contribution from proteins, carbohydrates, nucleic acids
6
2929
CH2 asymmetric stretching: mainly lipids, with a little contribution from proteins, carbohydrates, nucleic acids
7
2874
CH3 symmetric stretching: mainly proteins, with a little contribution from lipids, carbohydrates, nucleic acids
8
2855
CH2 symmetric stretching: mainly lipids, with a little contribution from proteins, carbohydrates, nucleic acids
9
1740
Ester C=O stretch: triacylglycerols, cholesterol esters
10
1656
Amide I (protein C=O stretching)
11
1540
Amide II (protein N-H bend, C-N stretch)
12
1452
CH2 bending: mainly lipids, with a little contribution from proteins
13
1392
COO− symmetric stretching: fatty acids
14
1343
CH2 side chain vibrations of collagen
15
1261
PO− 2 asymmetric stretching, non-hydrogen-bonded: mainly nucleic acids with a little contribution from
phospholipids
16
1236
PO− 2 asymmetric stretching, fully hydrogen-bonded: mainly nucleic acids with a little contribution from
phospholipids
17
1170
CO-O-C asymmetric stretching: ester bonds in cholesteryl esters
18
1080
PO− 2 symmetric stretching: nucleic acids and phospholipids C-O stretch: glycogen, polysaccharides,
glycolipids
19
1044
20
976
C-O stretching: polysaccharides
CN-C stretch: nucleic acids, ribose-phosphate main-chain vibrations of RNA
21
930
Z-type DNA
22
802
Vibrations in N-type sugars in nucleic acid backbone
Mann–Whitney U test. A P value less than or equal to 0.05 was
considered as statistically significant. The degree of significance
was denoted as *P < 0.05, **P < 0.01 and ***P < 0.001.
RESULTS AND DISCUSSION
The present study was carried out to determine simvastatininduced molecular variations in EDL muscle using FTIR spectroscopy. In this study, the EDL muscle was studied because it
is known to be more affected by statin therapy [18]. It is well
known that EDL muscle is largely composed of type II skeletalmuscle fibres (3 % type I, 57 % type IIa and 40 % type IIb) having
a fast-contracting glycolytic characteristic [19].
Simvastatin treatment led to a decrease in the weight of
drug-treated animals at the end of 1 month of treatment period
(P < 0.05). The initial weight of these animals was 273.33 +
−
12.72 g and at the end of the treatment period they weighed
246.83 +
− 12.90 g. On the other hand, the initial weight of the control animals was 258.37 +
− 10.84 g and at the end of the vehicle
administration period they weighed 269.87 +
− 17.04 g.
Simvastatin treatment at 20 mg/kg per day for 30 days significantly lowered the plasma cholesterol levels (P < 0.05). The
mean plasma cholesterol level of simvastatin-treated animals was
123.05 +
− 5.89 μg/ml, whereas it was 134.56 +
− 10.23 μg/ml in the
control animals.
In the present study, FTIR spectroscopy of dried tissue samples
was performed. This method was extensively used by our group
[13,20] and others [21,22] to study homogeneous tissue samples.
In the drying process, free and unbound water was removed from
the system, whereas intra- and inter-molecular water remained
in the system as can be seen in Table 1 and Figure 2(A) [20].
Since the free water was removed from the system, it may not
monitor well the properties of biological samples in the hydrated
state. As we have reported previously, there can be differences
in the IR spectra, especially between dissolved and freeze-dried
proteins [20]. It is also well known that the physical properties
of membranes vary as hydration level changes [23]. Furthermore, the removal of water from the specimens causes lyotropic
mesomorphic changes in lipid phase behaviour, which certainly
will cause some insignificant interpretations extrapolated to hydrated systems [24,25]. Therefore the studies with dry biological
samples are not appropriate for quantitative measurements, but instead can be used to deduce relative information. To achieve this,
we were very careful to perform the experiments under identical
..........................................................................................................................................................................................................................................................................................................................................................................
www.bioscirep.org / Volume 30 (1) / Pages 41–50
43
N. Simsek Ozek and others
Figure 1
Representative IR spectra of control EDL muscle in the 3750–750 cm−1 region
conditions, e.g. the same amount of dehydration, equivalent temperature and pressure. In the present study, since the main point
of interest was not to determine the properties of real hydrated
systems, but instead to determine the relative changes between
macromolecular composition and structure of two identical preparations of simvastatin-treated and untreated tissues, this may
not introduce a significant problem.
Figure 1 presents a representative IR spectrum of control EDL,
in the region of 3750–750 cm−1 . As can be seen from the Figure,
the FTIR spectrum of muscle is quite complex representing many
different functional groups of lipids, carbohydrates, proteins and
nucleic acids. Therefore a detailed spectral analysis was performed in three distinct regions, namely 3750–3025, 3025–2800
and 1900–780 cm−1 , which were given in Figures 2(A)–2(C) respectively. The position and assignments of the bands are listed
in Table 1. The changes in the frequency of these bands monitor structural variations; the signal intensity, or more accurately
the area under the bands, gives information about the concentration of the related molecules [14,15]. Although the normalized
average spectra were presented in the Figures to visually demonstrate the comparative changes, for the accurate determination
of variations in the spectral parameters, raw spectra belonging
to the control and simvastatin-treated samples were considered.
Baselined spectra were used only for band area determination.
The mean values and statistical analysis were performed, accordingly.
As can be seen from Figures 1 and 2(A), the broad band located
between 3600 and 3100 cm−1 mainly consists of two bands. The
band located at approx. 3500–3300 cm−1 mainly arises from
the N-H stretching vibrations of hydrogen-bonded amide groups
of proteins (amide A) with little contribution from the O-H
stretching of polysaccharides and intermolecular H-bonding
(band 2) [13,26]. The contribution of free water molecules to this
band can be neglected because free or unbound water was largely
removed during the preparation of the skeletal-muscle speci-
mens. This band overlaps with the O-H stretching (intramolecular H-bonding, which was labelled as band 1) located at approx.
3450–3570 cm−1 [13,27]. This band monitors the bound water in
skeletal-muscle samples.
In the C-H stretching region, absorptions arising from the
olefinic =CH, CH2 and CH3 stretching groups are monitored
(Figure 2B). The olefinic band (3010 cm−1 ) arises from the C-H
stretching mode of the HC=CH groups and can be used as a
measure of unsaturation in the acyl chains [12,28]. As seen from
the Figure, the area of this band decreased in simvastatin-treated
muscle (P < 0.05), indicating a decrease in the population of
unsaturation in acyl chains of lipid molecules. This loss of unsaturation may be due to an increase in lipid peroxidation [28].
It was previously reported that after the termination of statin
therapy, the level of 8-epi-PGF2a in serum, plasma and urine decreased, which is an indicator of a significant in vivo oxidation injury, supporting our result [29]. In addition, the frequency of this
band shifted to higher values in simvastatin-treated EDL muscle
(P < 0.01), indicating disordering of the unsaturated lipids [30].
The CH2 antisymmetric/symmetric and CH3 asymmetric stretching bands originate mainly from the lipid acyl chains (Table 1).
The area of these bands decreased (P < 0.05) in simvastatintreated muscle. This suggests a reduction in the amount of lipids
and a change in the composition of the acyl chains [15]. This result
was also supported by the decrease in the area of the CH2 bending
mode belonging mainly to lipids, at 1452 cm−1 , COO− symmetric stretching (fatty acids) at 1392 cm−1 and in the ratio of lipid
bands (CH2 asym./CH2 sym.). This prevalent decrease in
lipid content may be due to an increase in skeletal-muscle lipolysis or utilization of lipid sources because of statin treatment.
By monitoring the olefinic band, we cannot clearly distinguish
whether the decrease in this band is due to a decrease in the concentration of unsaturated lipids or due to change in unsaturation.
In the present study, we also obtained the ratio of saturated to unsaturated lipids by calculating the ratio of the areas of the bands
..........................................................................................................................................................................................................................................................................................................................................................................
44
C The
Authors Journal compilation
C 2010
Biochemical Society
Effects of low dose simvastatin on rat skeletal EDL muscle tissue
Figure 2
Representative IR spectra of control and simvastatin-treated EDL muscles
(A) The 3750–3025 cm−1 region; (B) the 3025–2800 cm−1 region (C-H stretching region); (C) the 1900–780 cm−1 region
(‘fingerprint’ region). The spectra were normalized with respect to the CH2 asymmetric stretching band (A, B) and the
amide I band (C).
..........................................................................................................................................................................................................................................................................................................................................................................
www.bioscirep.org / Volume 30 (1) / Pages 41–50
45
N. Simsek Ozek and others
Table 2 Changes in the frequency and area values of the IR bands for control and simvastatin-treated EDL muscles
The values are the means +
− S.D. for each group. Comparisons were done by the Mann–Whitney U-test. The downward arrow indicates a decrease
and the upward arrow indicates an increase with respect to the control. The degree of significance is denoted as *P < 0.05, **P < 0.01 and
***P < 0.001. a.u., arbitrary unit.
Band frequency (cm−1 )
Band no.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Band area (a.u. · cm−1 )
Control EDL (n = 10)
Simvastatin-treated EDL (n = 8)
Control EDL (n = 10)
Simvastatin-treated EDL (n = 8)
3337.88 +
− 11
3065.97 +
− 1.65
3009.98 +
− 0.36
2960.49 +
− 1.47
2927.48 +
− 0.64
2873.58 +
− 0.36
2854.41 +
− 0.26
1744.49 +
− 0.88
1655.85 +
− 0.73
1541.21 +
− 0.79
1451.34 +
− 0.60
1393.24 +
− 0.68
1343.15 +
− 0.75
1261.32 +
− 0.54
1237.92 +
− 1.21
1167.51 +
− 0.59
1082.13 +
− 1.37
1042.78 +
− 0.90
973.84 +
− 1.32
929.21 +
− 1.53
802.22 +
− 1.21
3345.89 +
− 24.80↑
3068.12 +
− 1.17*↑
3010.83 +
− 0.54**↑
2959.46 +
− 2.00↓
2926.53 +
− 0.52*↓
2873.70 +
− 0.32↑
2854.32 +
− 0.35↓
1746.04 +
− 1.13*↑
1656.69 +
− 0.70*↑
1541.92 +
− 0.51↑
1453.08 +
− 1.07**↑
1391.24 +
− 0.45↓
1344.62 +
− 0.94*↑
1262.02 +
− 0.64**↑
1238.22 +
− 0.52↑
1169.09 +
− 1.68↑
1084.85 +
− 1.47**↑
1042.10 +
− 0.71↓
975.40 +
− 0.90*↑
929.98 +
− 1.33↑
802.43 +
− 1.10↑
45.85 +
− 5.33
4.79 +
− 0.63
1.15 +
− 0.03
2.54 +
− 0.12
3.67 +
− 0.20
0.96 +
− 0.13
0.77 +
− 0.10
2.73 +
− 0.42
24.35 +
− 3.71
16.11 +
− 2.43
3.44 +
− 0.53
4.58 +
− 0.72
1.26 +
− 0.07
1.59 +
− 0.28
3.52 +
− 0.60
2.19 +
− 0.19
2.52 +
− 0.229
1.30 +
− 0.19
0.28 +
− 0.05
0.14 +
− 0.002
0.08 +
− 0.02
37.51 +
− 4.20*↓
3.72 +
− 0.51**↓
1.06 +
− 0.06*↓
2.31 +
− 0.20*↓
3.32 +
− 0.10*↓
0.50 +
− 0.09***↓
0.53 +
− 0.15↓
2.15 +
− 0.31*↓
17.26 +
− 2.94**↓
11.48 +
− 1.85**↓
2.82 +
− 0.36*↓
3.88 +
− 0.83↓
1.14 +
− 0.11*↓
1.05 +
− 0.20**↓
2.82 +
− 0.47*↓
1.83 +
− 023*↓
2.06 +
− 0.19**↓
0.21 +
− 0.19**↓
0.21 +
− 0.05**↓
0.09 +
− 0.02**↓
0.05 +
− 0.01*↓
arising from saturated lipids (CH2 asymmetric+CH2 symmetric)
and unsaturated lipids (=CH). A significant increase in this ratio
(P < 0.01) from 3.06 +
− 1.38 to 6.76 +
− 0.65 was observed, which
indicates a more pronounced decrease in the amount of unsaturated lipids in comparison with the saturated ones, since simvastatin treatment induces a decrease in the content of both types of
lipids [14]. It is known that free radicals modify the lipid composition of the membranes by altering the unsaturated/saturated
fatty acid ratio of membrane phospholipids. The increase in this
ratio indicates that simvastatin treatment might induce lipid peroxidation in tissues due to the formation of free radicals.
The shifts in the frequencies of the CH2 stretching vibrations can be used as markers for the detection of changes in
acyl chain flexibility (order/disorder state of lipids). As can be
seen from Table 2 and Figure 3, the peak positions of these
bands shifted slightly but significantly to lower values (P < 0.05).
This indicates an increase in the number of trans conformers of
lipid molecules, i.e. an increase in lipid order [32]. One possible explanation for the observed membrane rigidity can be
the simvastatin-induced domain formation since we recently
reported simvastatin-induced lateral phase separation in model
membranes using differential scanning calorimetry [33]. Another
explanation may be free radical formation as a result of statin
treatment. Free radicals modify the lipid composition of the
membranes by altering the unsaturated/saturated fatty acid ratio of membrane phospholipids. This results in an increase in the
rigidity of the membrane [31].
Bandwidths of the C-H bands give information about the dynamics of the system [30,34]. The increase in the bandwidth
of the CH2 asymmetric stretching band suggests an increase in
the lipid fluidity of the treated muscle (P < 0.01) [12]. We also
calculated the bandwidth values of the CH2 asymmetric stretching band from the well-resolved second derivative spectra and
found similar results to those obtained from the absorption signal
(Table 3). This may be due to a decrease in the cholesterol level
since membrane cholesterol concentration affects its fluidity [35].
It has also been reported that statins affect skeletal-muscle membrane fluidity through changes in cholesterol content [6,36]. We
indeed observed a dramatic decrease (P < 0.05) in the 1740 cm−1
band, which is due to cholesterol esters and triacylglycerols (Figure 2C), implying a relative decrease in the concentration of
ester groups belonging to triacylglycerols and cholesterol esters
[15,37]. This was also confirmed by the decline in the area of
the other cholesterol ester band, namely CO-O-C asymmetric
stretching bond located at 1170 cm−1 [38].
One of the important factors affecting the membrane structure and dynamics is the amount of proteins and lipids in
the membranes [14,15]. A precise lipid-to-protein ratio can be
derived from the FTIR spectrum by calculating the ratio of the
areas of the bands arising from lipids (CH2 asymmetric, CH2
symmetric), and proteins (amide I). As seen from Table 3, the
ratio (CH2 asym. + CH2 sym./amide I, CH2 asym. stretch. +
CH2 sym. stretch./CH3 sym. stretch.) was much higher in
simvastatin-treated EDL muscle (P < 0.01). The increase of this
..........................................................................................................................................................................................................................................................................................................................................................................
46
C The
Authors Journal compilation
C 2010
Biochemical Society
Effects of low dose simvastatin on rat skeletal EDL muscle tissue
Table 3 Changes in the bandwidth values and the area ratios of some of the IR bands for control and simvastatin-treated EDL muscles
The values are the means +
− S.D. for each group. Comparisons were done by the Mann–Whitney U-test. The downward arrow indicates a decrease
and the upward arrow indicates an increase with respect to the control. The degree of significance is denoted as *P < 0.05 and **P < 0.01.
Bandwidth value (cm−1 ) or area ratio
Functional group
Control EDL (n = 10)
Simvastatin-treated EDL (n = 8)
Bandwidth of CH2 asym. (absorption spectrum)
12.56 +
− 0.68
Bandwidth of CH2 asym. (second derivative spectrum)
12.59 + 1.67
13.39 +
− 0.76**↑
Bandwidth of amide I
44.82 +
− 0.55
43.41 +
− 0.89**↓
7.00 +
− 0.45*↑
0.43 +
− 0.09**↑
0.18 +
− 0.02 **↑
6.76 +
− 0.65 **↑
Ratio of areas of CH2 asym. stretch. + CH2 sym. stretch./CH3 sym. stretch.
CH2 asym./CH2 sym.
4.25 + 0.75
0.15 +
− 0.01
0.15 +
− 0.01
3.06 +
− 1.38
7.23 + 1.16
Amide I/amide II
1.51 + 0.006
Ratio of areas of CH2 asym. stretch. + CH2 sym. stretch./amide I
Ratio of areas of PO2 sym./amide II
Ratio of areas of CH2 asym. + CH2 sym./olefinic band
Figure 3
14.82 + 0.67**↑
4.34 + 0.58*↓
1.49 + 0.01*↓
Representative (A) absorbance IR spectra and (B) second derivative spectra of amide I band for control and
simvastatin-treated EDL muscles in the 1700–1620 cm−1 region
Vector normalization was performed in the 1700–1600 cm−1 region. Absorption maxima appear as minima in the second
derivatives.
..........................................................................................................................................................................................................................................................................................................................................................................
www.bioscirep.org / Volume 30 (1) / Pages 41–50
47
N. Simsek Ozek and others
Table 4 Changes in the intensities of the main protein secondary structures for control and simvastatin-treated EDL muscles
The values are the means +
− S.D. for each group. Comparisons were done by the Mann–Whitney U-test. The downward arrow indicates a decrease
and the upward arrow indicates an increase with respect to the control. The degree of significance is denoted as *P < 0.05 and **P < 0.01. a.u.,
arbitrary unit.
Secondary structure intensity (a.u.)
Peak number
Structure
Peak centres (cm−1 )
Control (n = 10)
Simvastatin-treated EDL (n = 8)
1
Antiparallel β-sheet
1690
2
Turns
1676
3
α-Helix
1652
4
Random coils
1642
5
β-Sheet
1638
6
Aggregated β-sheet
1625
−0.14 +
− 0.02
−0.07 +
− 0.01
−0.18 +
− 0.03
−0.15 +
− 0.02
−0.15 +
− 0.01
−0.07 +
− 0.01
−0.20 +
− 0.01**↑
−0.10 +
− 0.03↑
−0.20 +
− 0.02↑
−0.17 +
− 0.02*↑
−0.12 +
− 0.01*↓
−0.10 +
− 0.01*↑
ratio in the treated muscle suggests that there was a much more
pronounced decrease in protein content when compared with
those of lipid content.
The bands at approx. 1657 and 1541 cm−1 can be attributed
to amide I and II vibrations of structural proteins respectively
[39]. As can be clearly seen in Table 2, the significant decrease
in the area values of these bands indicates a reduction in the
protein content in treated muscle samples. These results were
also consistent with the decrease in other protein bands, namely
amide A band (at 3330 cm−1 ), amide B band (at 3062 cm−1 ),
CH3 symmetric stretching band (at 2876 cm−1 ) and in the ratio
of protein bands (amide I/amide II). A possible explanation for
the decrease in protein content is that statins decrease the serum
levels of lipids and fatty acids and this situation forces the muscle
to use amino acids from proteins as an energy source. It has
been suggested that extreme protein degradation will damage the
muscle if the usage of proteins by skeletal muscle continues for a
long period and this may cause the formation of acute myopathy
and rhabdomyolysis [40].
The bandwidth of amide I band decreased significantly
(P < 0.01) (Table 3) and the frequency of the amide I and II
bands shifted to higher values in simvastatin-treated EDL muscle
(Table 2), indicating conformational changes in tissue proteins
[15]. It was previously reported that in more than 50 % of
patients, biochemical and/or genetic abnormalities of proteins
or genes that are involved in skeletal-muscle energy metabolism are associated with statin-related myopathy, supporting the
changes in the synthesis and conformation of tissue proteins induced by statins [41]. The changes in the protein structure were
determined from the intensities of the sub-bands in the second
derivative of the amide I absorbance band where six peaks were
observed (Figure 3B) and the intensity values of them are listed
in Table 4. The peak located at 1676 cm−1 is due to antiparallel
β-sheet structure, the peak at 1680 cm−1 arises from turns and
bends, the peak located at 1638 cm−1 is due to β-sheet structures,
the peak at 1652 cm−1 corresponds to an α-helix structure, the
peak at 1642 cm−1 is assigned to random coil structure and
the peak at approx. 1625 cm−1 is attributed to aggregated βsheet structure [42]. The results revealed that simvastatin treatment significantly decreased β-sheet structure and significantly increased random coil, antiparallel and aggregated β-sheet
structure. The increase in random coil and aggregated β-sheet
structures indicates simvastatin-induced protein denaturation
[43,44].
As seen from Table 2 and Figure 2(C), the significant decrease
(P < 0.05) in the area of the band located at 1343 cm−1 , which
originates from CH2 side chain vibrations of collagen [45], indicates a decrease in the amount of collagen in simvastatin-treated
muscle. It has been previously reported that pravastatin inhibits
type IV collagen secretion in fetal calf serum because of suppression of mevalonate pathway products, which play a critical role
in the proliferation of mammalian cells [46].
The asymmetric and symmetric phosphate stretching bands
at 1261, 1236 and 1080 cm−1 respectively originate mainly due
to the phosphodiester backbone of cellular nucleic acids and
phospholipids [13,15]. As seen from Figure 2(C) and Table 2, the
frequencies of these bands shifted to higher values in simvastatintreated muscles (P < 0.01 at 1261 and 1080 cm−1 ). The shifts to
higher values in the frequencies of these bands indicate dehydration around the PO2 − functional groups in phospholipids and/or a
change in the conformation of nucleic acids. Moreover, the areas
of these bands decreased dramatically in EDL muscle (P < 0.05
at 1236 and 1080 cm−1 ). This decrease is consistent with the decrease at 976, 929 and 806 cm−1 bands, which are also due to
RNA and DNA. These results imply a decrease in the relative concentration of the nucleic acids in the treated muscle, which may
be due to statin-induced nuclear condensation and fragmentation
through the activation of caspase 3 enzymes [47].
The peak area ratio of the bands at 1080 cm−1 (phosphate symmetric stretching nucleic acid) and 1540 cm−1 (amide II-protein)
is often used to illustrate the change in the DNA and protein
content of cells [48]. A decrease in both protein and DNA content was observed in simvastatin-treated EDL muscle (Table 2).
A higher ratio value implies a more dramatic decrease in protein content when compared with the DNA content (P < 0.01)
(Table 3). Our result supported that statins affect the ubiquitin
proteasome pathway which is tightly regulated and responsible
for the recognition and degradation of most of the proteins in
skeletal muscle [49].
As seen from the Tables, the spectral values between the control and simvastatin-treated groups seem to be small, but the
changes are so consistent that the S.D. values are very small and
significant, which again shows the high sensitivity of the FTIR
spectroscopy technique. In FTIR spectroscopy, the data obtained
..........................................................................................................................................................................................................................................................................................................................................................................
48
C The
Authors Journal compilation
C 2010
Biochemical Society
Effects of low dose simvastatin on rat skeletal EDL muscle tissue
are stored in digitally encoded formats, which facilitate spectral
interpretation with the aid of post-acquisition data manipulation
algorithms. This property of the technique allows the accurate
detection of small changes even in the weak absorption bands
[12].
In conclusion, in the present study, by using FTIR spectroscopy, it was observed that simvastatin treatment induced significant structural, functional and compositional alterations in
the rat EDL muscles. A decrease was found in the amount of
protein, saturated and unsaturated lipids, cholesterol esters, triacylglycerols, glycogen and nucleic acid in simvastatin-treated
EDL muscles. Simvastatin treatment also leads to a conformational change in protein and nucleic acid structure. In addition, in
the present study, information on the effect of simvastatin on lipid
order, fluidity and protein secondary structure was reported for the
first time, in skeletal muscle. It was observed that simvastatin increased lipid order and fluidity. While the fraction of random coil,
antiparallel and aggregated β-sheet structure increased, β-sheet
structure decreased indicating simvastatin-induced protein denaturation. These kinds of structural, compositional and dynamical
information are very important, because today a correlation of
these parameters to ion channel activities has been proposed. It
is known that any change in these parameters causes alterations
in the function of ion channels [50].
FUNDING
8
9
10
11
12
13
14
15
16
This work was supported by Middle East Technical University–State
Planning Organization Research Fund [grant number BAP-08-11DPT2002K120510-TB-04].
17
REFERENCES
18
19
1 Evans, M. and Rees, A. (2002) Effects of HMG-CoA reductase
inhibitors on skeletal muscle: are all statins the same.
Curr. Opin. Lipidol. 13, 415–420
2 Dirks, A. J. and Jones, K. M. (2006) Statin-induced apoptosis and
skeletal myopathy. Am. J. Physiol. Cell Physiol. 291, C1208–C1212
3 Brown, M. S. and Goldstein, J. L. (2004) Lowering plasma
cholesterol by raising LDL receptors. Atheroscler. Suppl. 5, 57–59
4 de Lemos, J. A., Blazing, M. A., Wiviott, S. D., Lewis, E. F., Fox,
K. A. A., White, H. D., Rouleau, J. L., Pedersen, T. R., Gardner,
L. H., Mukherjee, R. et al. (2004) Early intensive vs a delayed
conservative simvastatin strategy in patients with acute coronary
syndromes: phase Z of the A to Z trial. JAMA, J. Am. Med. Assoc.
292, 1307–1316
5 Masters, B. A., Palmowski, M. J., Flint, O. P., Gregg, R. E.,
Wang-Iverson, D. and Durham, S. K. (1995) In vitro myotoxicity of
the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors,
pravastatin, lovastatin, and simvastatin, using neonatal rat
myocytes. Toxicol. Appl. Pharmacol. 131, 163–174
6 Baker, S. K. and Tarnopolsky, M. A. (2001) Statin myopathies:
pathophysiologic and clinical perspectives. Clin. Invest. Med. 24,
258–272
7 Morita, I., Sato, I., Ma, L. and Murota, S. (1997) Enhancement of
membrane fluidity in cholesterol poor endothelial cells pre-treated
with simvastatin. Endothelium 5, 107–113
20
21
22
23
24
25
26
Cziraky, M. J., Willey, V. J., McKenney, J. M., Kamat, S. A., Fisher,
M. D., Guyton, J. R., Jacobson, T. A. and Davidson, M. H. (2006)
Statin safety: an assessment using an administrative claims
database. Am. J. Cardiol. 97, S61–S68
Schick, B. A., Laaksonen, R., Frohlich, J. J., Päivä, H.,
Lehtimäki, T., Humphries, K. H. and Côté, H. C. F. (2007)
Decreased skeletal muscle mitochondrial DNA in patients treated
with high-dose simvastatin. Clin. Pharmacol. Ther. 81, 650–653
Phillips, P. S., Haas, R. H., Bannykh, S., Hathaway, S., Gray, N. L.,
Kimura, B. J., Vladutiu, G. D. and England, J. D. (2002)
Statin-associated myopathy with normal creatine kinase levels.
Ann. Intern. Med. 137, 581–585
Yokoyama, M., Seo, T., Park, T., Yagyu, H., Hu, Y., Son, N. H.,
Augustus, A. S., Vikramadithyan, R. K., Ramakrishnan, R., Pulawa,
L. K. et al. (2007) Effects of lipoprotein lipase and statins on
cholesterol uptake into heart and skeletal muscle. J. Lipid Res.
48, 646–655
Toyran, N., Lasch, P., Naumann, D., Turan, B. and Severcan, F.
(2006) Early alterations in myocardia and vessels of the diabetic
rat heart: an FTIR microspectroscopic study. Biochem. J. 397,
427–436
Severcan, F., Gokduman, K., Dogan, A., Bolay, S. and Gokalp, S.
(2008) Effects of in-office and at-home bleaching on human
enamel and dentin: an in vitro application of FTIR study.
Appl. Spectrosc. 62, 1274–1279
Dogan, A., Ergen, K., Budak, F. and Sevecan, F. (2007) Evaluation
of disseminated candidiasis on an experimental animal model: a
Fourier transform infrared study. Appl. Spectrosc. 61, 199–203
Cakmak, G., Togan, I. and Severcan, F. (2006) 17β-Estradiol
induced compositional, structural and functional changes in
rainbow trout liver, revealed by FT-IR spectroscopy: a comparative
study with nonylphenol. Aquat. Toxicol. 77, 53–63
Naumann, D. (2001) FT-infrared and FT-Raman spectroscopy in
biomedical research. Appl. Spectrosc. Rev. 36, 239–298
Yilmaz, O., Keser, S., Tuzcu, M. and Çetintaş, B. (2007)
Resveratrol (trans-3,4 ,5-trihydroxystilbene) decreases lipid
peroxidation level and protects antioxidant capacity in sera and
erythrocytes of old female Wistar rats induced by the kidney
carcinogen potassium bromate. Environ. Toxicol. Pharmacol. 24,
79–85
Westwood, F. R., Bigley, A., Randall, K., Marsden, A. M. and Scott,
R. C. (2005) Statin-induced muscle necrosis in the rat: distribution,
development, and fibre selectivity. Toxicol. Pathol. 33, 246–257
Delp, M. D. and Duan, C. J. (1996) Composition and size of type I,
IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle.
Appl. Physiol. 80, 261–270
Akkas, S. B., Severcan, M., Yilmaz, O. and Severcan, F. (2007)
Effects of lipoic acid supplementation on rat brain tissue: an FTIR
and neural network study. Food Chem. 105, 1281–1288
Crupi, V., De Domenico, D., Interdonato, S., Majolino, D., Maisano,
G., Migliardo, P. and Venuti, V. (2001) FT-IR spectroscopy study on
cutaneous neoplasie. J. Mol. Struct. 563–564, 115–118
Jagadeesan, G., Kavitha, A. V. and Subashini, J. (2005) FT-IR study
of the influence of Tribulus terrestris on mercury intoxicated mice,
Mus musculus liver. Trop. Biomed. 22, 15–22
Jay Mashl, R., Larry Scott, H., Subramaniam, S. and Jakobsson, E.
(2001) Molecular simulation of dioleoylphosphatidylcholine lipid
bilayers at differing levels of hydration. Biophys. J. 81, 3005–3015
Beney, L., Mille, Y. and Gervais, P. (2004) Death of Escherichia coli
during rapid and severe dehydration is related to lipid phase
transition. Appl. Microbiol. Biotechnol. 65, 457–464
Silver, B. L. (1985) The Physical Chemistry of Membranes: An
Introduction to the Structure and Dynamics of Biological
Membranes, pp. 40–46, Allen and Unwin, Boston
Yang, J. and Yen, H. E. (2002) Early salt stress effects on the
changes in chemical composition in leaves of ice plant and
arabidopsis. A Fourier transform infrared spectroscopy study.
Plant Physiol. Preview 130, 1032–1042
..........................................................................................................................................................................................................................................................................................................................................................................
www.bioscirep.org / Volume 30 (1) / Pages 41–50
49
N. Simsek Ozek and others
27 Evans, L. A., Macey, D. J. and Webb, J. (1990) Characterization
and structural organization of the organic matrix of the radula teeth
of the chiton Acanthopleura hirtosa. Phil. Trans. R. Soc. Lond. B,
Biol. Sci. 329, 87–96
28 Severcan, F., Gorgulu, G., Gorgulu, S. T. and Guray, T. (2005) Rapid
monitoring of diabetes-induced lipid peroxidation by Fourier
transform infrared spectroscopy: evidence from rat liver
microsomal membranes. Anal. Biochem. 339, 36–40
29 Sinzinger, H., Lupattelli, G. and Chehne, F. (2000) Increased lipid
peroxidation in a patient with CK-elevation and muscle pain during
statin therapy. Atherosclerosis 153, 255–256
30 Lopez-Garcia, F., Villian, J. and Gomez-Fernandez, J. C. (1993)
Infrared spectroscopic studies of the interaction of diacylglycerols
with phosphatidylglycerol, phosphatidylserine in the presence of
calcium. Biochim. Biophys. Acta 1169, 264–272
31 Curtis, M. T., Gilfor, D. and Farber, J. L. (1984) Lipid peroxidation
increases the molecular order of microsomal membranes.
Arch. Biochem. Biophys. 235, 644–649
32 Severcan, F. (1997) Vitamin E decreases the order of the
phospholipid model membranes in the gel phase: an FTIR study.
Biosci. Rep. 17, 231–235
33 Kocak, M. and Severcan, F. (2007) Interactions of cholesterol
reducing agent simvastatin with neutral DPPC and DMPC
multilamellar liposomes. 2007 Biophysical Society Meeting
Abstracts, Biophysical Journal, Supplement, 59a, Abstract,
270-Pos
34 Schultz, C. and Naumann, D. (1991) In vivo study of the state of
order of the membranes of Gram-negative bacteria by Fouriertransform infrared spectroscopy (FT-IR). FEBS Lett. 294, 43–46
35 Vevera, J., Fisar, Z., Kvasnicka, T., Zdenek, H., Starkova, L.,
Ceska, R. and Papezova, H. (2005) Cholesterol-lowering therapy
evokes time-limited changes in serotonergic transmission.
Psychiatry Res. 133, 197–203
36 Wortmann, R. L. (2002) Lipid-lowering agents and myopathy.
Curr. Opin. Rheumatol. 14, 643–647
37 Nara, M., Okazaki, M. and Kagi, H. (2002) Infrared study of human
serum very low-density and low-density lipoproteins. Implication of
esterified lipid C=O stretching bands for characterizing
lipoproteins. Chem. Phys. Lipids 117, 1–6
38 Jackson, M., Ramjiawan, B., Hewko, M. and Mantsch, H. H. (1998)
Infrared microscopic functional group mapping and spectral
clustering analysis of hypercholesterolomic rabbit liver.
Cell. Mol. Biol. 44, 89–98
39 Manoharan, R., Baraga, J. J., Rava, R. P., Dasari, R. R.,
Fitzmaurice, M. and Feld, M. S. (1993) Biochemical analysis and
mapping of atherosclerotic human artery using FTIR
microspectroscopy. Atherosclerosis 103, 181–193
40 Motojima, K., Passilly, P., Peters, J. M., Gonzalez, F. J. and
Latruffe, N. (1998) Expression of putative fatty acid transporter
genes are regulated by peroxisome proliferator-activated receptor
alpha and gamma activators in a tissue- and inducer-specific
manner. J. Biol. Chem. 273, 16710–16714
41 Vladutiu, G. D., Simmons, Z., Isackson, P. J., Tarnopolsky, M.,
Peltier, W. L., Barboi, A. C., Sripathi, N., Wortmann, R. L. and
Phillips, P. S. (2006) Genetic risk factors associated with
lipid-lowering drug-induced myopathies. Muscle Nerve 34,
153–162
42 Lopez, G., Martinez, R., Gallego, J., Tarancon, M. J., Carmona, P.
and Fraile, M. V. (2001) Dietary fats affect rat plasma lipoprotein
secondary structure as assessed by Fourier transform infrared
spectroscopy. J. Nutr. 131, 1898–1902
43 Severcan, F. and Haris, P. (2003) Fourier transform infrared
spectroscopy suggests unfolding of loop structures precedes
complete unfolding of pig citrate synthase. Biopolymers 69,
440–447
44 Ellepola, S. W., Choi, S. M. and Ma, C. Y. (2005) Conformational
study of globulin from rice (Oryza sativa) seeds by Fouriertransform infrared spectroscopy. Int. J. Biol. Macromol. 37,
12–20
45 Bi, X., Yang, X., Bostrom, M. P. G., Bartusiak, D., Ramaswamy, S.,
Fishbein, K. W., Spencer, R. G. and Pleshko Camacho, N. (2007)
Fourier transform infrared imaging and MR microscopy studies
detect compositional and structural changes in cartilage in a
rabbit model of osteoarthritis. Anal. Bioanal. Chem. 387,
1601–1612
46 Nishimura, M., Tanaka, T., Yasuda, T., Kurakata, S., Kitagawa, M.,
Yamada, K., Saito, Y. and Hirai, A. (1999) Effect of pravastatin on
type IV collagen secretion and mesangial cell proliferation.
Kidney Int. Suppl. 71, S97–S100
47 Kubota, T., Fujisaki, K., Itoh, Y., Yano, T., Sendo, T. and Oishi, R.
(2004) Apoptotic injury in cultured human hepatocytes induced by
HMG-CoA reductase inhibitors. Biochem. Pharmacol. 67,
2175–2186
48 Mourant, J. R, Yamada, Y. R., Carpenter, S., Dominique, L. R. and
Freyer, J. P. (2003) FTIR spectroscopy demonstrates biochemical
differences in mammalian cell cultures at different growth stages.
Biophys. J. 85, 1938–1947
49 Urso, M. L., Clarkson, P. M., Hittel, D., Hoffman, E. P. and
Thompson, P. D. (2005) Changes in ubiquitin proteasome pathway
gene expression in skeletal muscle with exercise and statins.
Arterioscler. Thromb. Vasc. Biol. 25, 2560–2566
50 Awayda, M. S., Shao, W., Guo, F., Zeidel, M. and Hill, W. G. (2004)
ENaC membrane interactions: regulation of channel activity by
membrane order. J. Gen. Physiol. 123, 709–727
Received 1 October 2008/2 February 2009; accepted 19 February 2009
Published as Immediate Publication 19 February 2009, doi 10.1042/BSR20080150
..........................................................................................................................................................................................................................................................................................................................................................................
50
C The
Authors Journal compilation
C 2010
Biochemical Society