Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal muscle

49 Pages·2017·1.05 MB·English

Statin or fibrate chronic treatment modifies the
proteomic profile of rat skeletal muscle
Giulia Maria Camerino, Maria Antonietta Pellegrino, Lorenza Brocca,
Claudio Digennaro, Diana Conte Camerino, Sabata Pierno, Roberto Bottinelli
To cite this version:
Giulia Maria Camerino, Maria Antonietta Pellegrino, Lorenza Brocca, Claudio Digennaro, Diana
Conte Camerino, et al.. Statin or fibrate chronic treatment modifies the proteomic profile of rat
skeletal muscle. Biochemical Pharmacology, 2011, ￿10.1016/j.bcp.2011.01.022￿. ￿hal-00681625￿
HAL Id: hal-00681625
https://hal.science/hal-00681625
Submitted on 22 Mar 2012
HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est
archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents
entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non,
lished or not. The documents may come from émanant des établissements d’enseignement et de
teaching and research institutions in France or recherche français ou étrangers, des laboratoires
abroad, or from public or private research centers. publics ou privés.
Accepted Manuscript
Title:Statinorfibratechronictreatmentmodifiesthe
proteomicprofileofratskeletalmuscle
Authors:GiuliaMariaCamerino,MariaAntoniettaPellegrino,
LorenzaBrocca,ClaudioDigennaro,DianaConteCamerino,
SabataPierno,RobertoBottinelli
PII: S0006-2952(11)00080-3
DOI: doi:10.1016/j.bcp.2011.01.022
Reference: BCP10819
Toappearin: BCP
Receiveddate: 15-11-2010
Reviseddate: 27-1-2011
Accepteddate: 31-1-2011
Please cite this article as: Camerino GM, Pellegrino MA, Brocca L, Digennaro C,
Camerino DC, Pierno S, Bottinelli R, Statin or fibrate chronic treatment modifies
the proteomic profile of rat skeletal muscle, Biochemical Pharmacology (2010),
doi:10.1016/j.bcp.2011.01.022
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
Themanuscriptwillundergocopyediting,typesetting,andreviewoftheresultingproof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
applytothejournalpertain.
*Clean Manuscript

Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal
1
2
muscle
3
4
5

6
7
8 Giulia Maria Camerinoa, Maria Antonietta Pellegrinob, Lorenza Brocctab, Claudio
9
p
10
Digennaroa, Diana Conte Camerinoa, Sabata Piernoa1*, Roberto Bottinellib1.
11
12 i
r
13
14 c
15

16 s
17
18 a Department of Pharmacobiology, Section of Pharmacology, Facultuy of Pharmacy, University of
19
20
n
21 Bari “Aldo Moro”, Via Orabona 4, 70124, Bari, ITALY.
22
a
23
b Department of Physiology and Interuniversity Institute of Myology, University of Pavia, Via
24
25 M
26 Forlanini 6, 27100 Pavia, ITALY.
27
28

29
d
30
31
e
32
33
34 1These authors contributed equally tot this work as senior authors.
35 p
36

37
e
38
39 *Corresponding author at: Department of Pharmacobiology, Section of Pharmacology, Faculty of
c
40
41 Pharmacy, University of Bari “Aldo Moro”, Via Orabona 4, 70124, Bari, ITALY.
c
42
43
E-mail address: [email protected] (Sabata Pierno).
44
45
46
47
48

49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
1
64 Page 1 of 47
65
Abstract
1
2
3
4
Statins and fibrates can cause myopathy. To further understand the causes of the damage we
5
6
7 performed a proteome analysis in fast-twitch skeletal muscle of rats chronically treated with
8
t
9
different hypolipidemic drugs. The proteomic maps were obtained from extensor dipgitorum longus
10
11
12 (EDL) muscles of rats treated for 2-months with 10mg/kg atorvastatin, 2i0mg/kg fluvastatin,
r
13
14 60mg/kg fenofibrate and control rats. The proteins differentially expressedc were identified by mass
15
16 s
spectrometry and further analysed by immunoblot analysis. We found a significant modification in
17
18 u
19 40 out of 417 total spots analysed in atorvastatin treated rats, 15 out of 436 total spots in fluvastatin
20
n
21
treated rats and 21 out of 439 total spots in fenofibrate treated rats in comparison to controls. All
22
a
23
24 treatments induced a general tendency to a down-regulation of protein expression; in particular,
25 M
26
atorvastatin affected the protein pattern more extensively with respect to the other treatments.
27
28

29 Energy production systems, both oxidative and glycolytic enzymes and creatine kinase, were down-
d
30
31
regulated following atorvastatin administration, whereas fenofibrate determined mostly alterations
e
32
33
34 in glycolytic enzymes and creatine ktinase, oxidative enzymes being relatively spared. Additionally,
35 p
36 all treatments resulted in some modifications of proteins involved in cellular defenses against
37
e
38
oxidative stress, such as heat shock proteins, and of myofibrillar proteins. These results were
39
c
40
41 confirmed by immunoblot analysis. In conclusions, the proteomic analysis showed that either statin
c
42
43
or fibrate adminisAtration can modify the expression of proteins essential for skeletal muscle function
44
45
46 suggesting potential mechanisms for statin myopathy.
47
48

49
50
51
52
53 Keywords: hypolipidemic drugs, skeletal muscle, side effects, myopathy, proteomic analysis
54
55

56
57
58
59
60

61
62
63
2
64 Page 2 of 47
65
1. Introduction
1
2
3
4
Hypocholesterolemic drugs, such as statin and fibrate, are widely prescribed medications effective
5
6
7 to reduce blood lipids level. Statins potently inhibit the 3-hydroxymethyl-glutaryl-coenzyme A
8
t
9
(HMG-CoA) reductase, the rate-limiting enzyme for the synthesis of mevalonatep and therefore
10
11
12 cholesterol [1]. Fibrates by acting on the peroxisome proliferator-activated ireceptor (PPAR)-α,
r
13
14 lower serum triglyceride levels and increase high-density lipoprotein (HDL)c-cholesterol [2].
15
16 s
Statins and fibrate are generally well tolerated by patients, however they can potentially produce
17
18 u
19 serious adverse effects especially on skeletal muscle. The clinical evidence of statin-associated
20
n
21
muscle disorders range from benign myalgia to severe myopathy with elevation of serum creatine
22
a
23
24 kinase (CK) and muscle weakness. Life-threatening rhabdomyolysis with muscle necrosis and
25 M
26
electrolyte alteration, myoglobinuria and renal failure is extremely rare [3-5]. A population-based
27
28

29 study describes that the relative risk of myopathy associated with the use of fibrates in monotherapy
d
30
31
is 5-fold higher compared with statin [6]. However, the risk of rhabdomyolysis with cerivastatin
e
32
33
34 monotherapy was 10-fold greater thtan with other statins, and in combination with gemfibrozil, was
35 p
36 increased more than 1400-fold [7-9]. Thus, the fear of rare but serious muscle toxicity remains a
37
e
38
major impediment to the appropriate use of these drugs considering the ample number of patients
39
c
40
41 worldwide now receiving statins for hypercholesterolemia. In addition statins exhibit anti-
c
42
43
inflammatory andA antineoplastic pleiotropic effects that may expand their clinical value as well as
44
45
46 the need to identify the mechanism of muscle side effects and possibly to find suitable
47
48
countermeasures [10-12]. Indeed, the precise molecular mechanisms behind statin-associated
49
50
51 myopathy have not been fully elucidated, although various hypotheses suggested either the
52
53 alteration of muscle cell membrane function due to impairment of cholesterol synthesis, the deficits
54
55
in energy metabolism associated with ubiquinone deficiency [13] or the reduction of small GTP-
56
57
58 binding proteins involved in myocytes preservation [14]. Different studies have shown that statin
59
60
and fibrate can modify gene and protein expression in skeletal muscle [15]. For instance, an up-
61
62
63
3
64 Page 3 of 47
65
regulation of ryanodine receptor, suggestive of intracellular calcium increase, was found in muscle
1
2 biopsies of statin treated patients showing evident structural damage [16]. Our previous studies
3
4
have demonstrated that statin and fibrate affect skeletal muscle function also by modifying calcium
5
6
7 homeostasis and resting chloride conductance (gCl). Indeed, lipophilic statin increased intracellular
8
t
9
calcium via mitochondria and sarcoplasmic reticulum release [17], thereby affecpting contractile
10
11
12 function. In turn these drugs reduced resting gCl [18-19], a parameter sustaiined by the ClC-1
r
13
14 chloride channel and modulated by calcium-dependent PKC [20-25]. Thics parameter is normally
15
16 s
high in fast-twitch muscles and is important to guarantee muscle membrane potential and
17
18 u
19 excitability [20-22]. We also found that fenofibrate directly inhibit the ClC-1 chloride channel [20].
20
n
21
To better understand the mechanisms underlying myotoxicity and the diverse effects of statin and
22
a
23
24 fibrate, we applied the proteomic approach to analyse skeletal muscle adaptation to chronic drug
25 M
26
treatment. Since statins inhibit many enzymatic reactions downstream of mevalonate production,
27
28

29 they may affect a wide range of intracellular functions. Also fenofibrate by acting on peroxisome
d
30
31
proliferator-activated receptors (PPARs) has pleiotropic biological effects. In addition, skeletal
e
32
33
34 muscle adaptation is per se very comtplex because it depends on the activation of genetic programs
35 p
36 codifying for proteins belonging to different functional categories [26]. This widens the panorama
37
e
38
of potential sites of antilipidemic drug side effects and justifies the difficulties in understanding the
39
c
40
41 mechanisms of myotoxicity. Therefore, since complex modifications of the protein pattern are
c
42
43
likely to occur foAllowing both statin and fibrate administration, we believe that a global proteomic
44
45
46 approach may prove very helpful to clarify the molecular mechanisms underlying myopathy. Thus,
47
48
the proteomic analysis, by identifying differentially expressed protein following drug administration
49
50
51 appears the approach of choice to address the complexity of muscle adaptations and to reveal the
52
53 underlying mechanisms in statin and fibrate muscle damage. Despite the high resolutive power of
54
55
the 2DE approach and the large number of proteins whose differential expression could be studied
56
57
58 (~450), not all the protein expressed in the muscle could be examined, such as those with an
59
60
isoelectric point higher than 8-9 and little represented proteins such as membrane proteins.
61
62
63
4
64 Page 4 of 47
65
However, this approach has been demonstrated widely useful in a number of recent works [27-30].
1
2 Such a proteomic approach is novel in skeletal muscle. The few proteomic studies reported in the
3
4
literature have regarded other tissues, such as liver and interestingly showed that statin treatment
5
6
7 can modify the expression of the cholesterol biosynthesis pathway enzymes as well as of
8
t
9
carbohydrate metabolism enzymes, cellular stress proteins and protein involvped in calcium
10
11
12 homeostasis [31-32]. Statin by inhibiting cholesterol biosynthesis may alsoi affect glutathione
r
13
14 peroxidase liver production, whose expression level and catalytic activcity were reduced. The
15
16 s
consequence of this loss may be an increased sensitivity of the cells to peroxide [33].
17
18 u
19 Here we report the effects of chronic treatments with fluvastatin, atorvastatin and fenofibrate in rats
20
n
21
on the proteomic maps of the extensor digitorum longus (EDL) muscle. Although there are some
22
a
23
24 reports showing that fast-twitch muscles are primarily affected in statin myopathy [34], whereas
25 M
26
type I muscle fibers are mainly affected by fibrates [35], we focused the proteomic analysis on the
27
28

29 fast-twitch EDL muscle because we previously showed that this muscle is a toxicological target of
d
30
31
both statins and fibrates [20]. The results reveal various metabolic pathways involved in the
e
32
33
34 response to antilipidemic drugs, whitch may concur to myotoxicity.
35 p
36
37
e
38

39
c
40
41
c
42
43
A
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
5
64 Page 5 of 47
65
2. METHODS
1
2
3
4
2.1. Animal care and treatments
5
6
7 The animal study protocol was conducted in accordance with the Italian Guidelines for the use of
8
t
9
laboratory animals, which conforms to the European Community Directive pubplished in 1986
10
11
12 (86/609/EEC). Adult male Wistar rats (Charles River Laboratories, Calco, Italy)i, weighing 300-350
r
13
14 g, were housed individually in appropriate metabolic cages in an environmcentally controlled room
15
16 s
and received commercial rodent chow (30 g/day) (Charles River, 4RF21) and water ad libitum. Rats
17
18 u
19 were randomly assigned to 4 experimental groups of 8 animals each: 1) fluvastatin 20mg/kg/day
20
n
21
treated animals (FLUVA), 2) atorvastatin 10mg/kg/day treated animals (ATO), 3) fenofibrate 60
22
a
23
24 mg/kg/day treated animals (FENO), 4) control animals (CTRL) treated with the vehicle (0.5%
25 M
26
carboxymethylcellulose in aqueous solution) used to dissolve the drugs. Fluvastatin (Lescol,
27
28

29 Novartis), atorvastatin (Torvast, Pfizer), and fenofibrate (Lipsin, Caber) were administered orally
d
30
31
by using an esophageal cannula, once a day for two months [20]. During the treatment the body
e
32
33
34 weight and vital parameters (healtht conditions, water and food consumption) were normal in all
35 p
36 treated rats. Skeletal muscle performance was evaluated daily by testing in each rat the righting
37
e
38
reflex, i.e. the ability of the rat to straighten itself on four legs when turned on the back. The
39
c
40
41 observation of the righting reflex can help to detect severe myotonic-like signs or alteration of
c
42
43
muscle function. AAs previously observed, the righting reflex was normal during the entire treatment
44
45
46 period in all the animals. No mortality was observed. At the end of the 2-months treatment rats were
47
48
sacrificed by cervical dislocation and the extensor digitorum longus (EDL) muscle was carefully
49
50
51 dissected from each rat and immediately frozen in liquid nitrogen, then stored at -80°C until
52
53 proteomic analysis. The contralateral EDL muscle of all fluvastatin, atorvastatin and fenofibrate
54
55
treated rats was immediately placed in a appropriate muscle bath chamber to measure the resting
56
57
58 chloride conductance (gCl) by the 2-intracellular microelectrode technique [20]. Histological
59
60
61
62
63
6
64 Page 6 of 47
65
analysis was also performed on four tibialis anterior muscles dissected from randomly selected
1
2 treated rats of each experimental group as previously described [24].
3
4

5
6
7 2.2. Proteome analysis (2-DE)
8
t
9
2.2.1. Sample preparation. The methods of proteome analysis are mostly the psame as those
10
11
12 previously used [29]. Muscle samples previously stored at -80°C, were pulverizied in a steel mortar
r
13
14 with liquid nitrogen to obtain a powder that was immediately resuspendecd in a lysis buffer [8M
15
16 s
urea, 2M thiourea, 4% CHAPS, 65mM DTT, 40mM Tris base (Healthcare, Germany) and a cocktail
17
18 u
19 of protease inhibitors (Sigma-Aldrich, Italy)]. The samples were vortexed, frozen with liquid
20
n
21
nitrogen and thawed at room temperature four times; then the samples were incubated with DNase
22
a
23
24 and RNase for 45 min at 4°C to separate proteins from nucleic acids and finally spun at 35000 g for
25 M
26
30 min. Protein concentration in the dissolved samples was determined with a protein assay kit (2D
27
28

29 quant Kit, Healthcare). In order to perform proteome analysis, a sample mix was obtained for each
d
30
31
experimental group (CTRL, FLUVA, ATO, FENO). Each sample mix contained an equal protein
e
32
33
34 quantity taken from each muscle samtple of CTRL, FLUVA, ATO, FENO.
35 p
36
37
e
38
2.2.2. Two-dimensional electrophoresis. Isoelectrofocusing was carried out using IPGphor system
39
c
40
41 (Ettan IPGphor isoelectric Focusing Sistem - Healthcare). IPG gels strips, pH 3-11 NL (non linear)
c
42
43
13 cm, were rehyAdrated for 14h, at 30 Volt and at 20°C, in 250 μl of reswelling buffer [(8 M urea, 2
44
45
46 M thiourea, 2% (w/v) CHAPS, 0.1% (v/v) tergitol NP7 (Sigma, Italy), 65 mM DTT, 0.5% (v/v)
47
48
pharmalyte 3-11NL (Healthcare), tergitol NP7 (Sigma)] containing 100 g protein sample. Strips
49
50
51 were focused at 20000 Vhr, at constant temperature of 20°C and the current was limited to 50 A°
52
53
per IPG gel strip. After isoelectrofocusing the strips were stored at -80°C until use or equilibrated
54
55
56 immediately for 10-12 min in 5 ml of equilibration buffer [50 mM Tris pH 6.8, 6 M urea, 30% (v/v)
57
58
glycerol, 2% (w/v) SDS, 3% (w/v) iodoacetamide (Healthcare)]. Then, the immobiline IPG gel
59
60
61 strips were applied to 15% SDS-PAGE without a stacking gel. The separation was performed at 80
62
63
7
64 Page 7 of 47
65
V for 17h at room temperature. The 2D gels were fixed for 2h in fixing solution [(ethanol 40% (v/v)
1
2 acetic acid 10% (v/v) (VWR International, Italy)], stained with fluorescent staining (FlamingoTM
3
4
Fluorescent Gel Stain by BIO-RAD, Italy) for 3h and destained with 0.1% (w/v) Tween 20 (VWR
5
6
7 International) solution for 10 minutes. Triplicate gels of each mix sample were obtained, visualized
8
t
9
using a Typhoon laser scanner (Healthcare) and analyzed with Platinum Software (Hpealthcare). The
10
11
12 analysis was performed comparing FLUVA, ATO and FENO with CTRL. Foir each analysis one
r
13
14 gel was chosen as the master gel, and used for the automatic matching of spcots in the other 2D gels.
15
16 s
Only spots present in all gels used for the analysis were considered. The software provided
17
18 u
19 normalized volume for each spot (representing protein amount). A good reproducibility of the spots
20
n
21
among the triplicate gels of each experimental group was found. Plotting the spot volumes for
22
a
23
24 matched spots on a linear scale, in fact, regression analysis yielded correlation coefficients in the
25 M
26
range 0.75-0.8. The volumes of each spots in the triplicate gels were averaged and the average
27
28

29 volume used for statistical comparison among spots was considered significant if P < 0.05. The
d
30
31
average volumes of each differentially expressed spot were used to determine the volume ratios
e
32
33
34 reported in the figures and tables. t
35 p
36
37
e
38
2.3. Protein identification
39
c
40
41 2.3.1. Electrophoresis fractionation and in situ digestion. 2D gels were loaded with 300 µg of
c
42
43
proteins per stripA and the electrophoretic was carried out with the same conditions described above.
44
45
46 After staining with Colloidal Coomassie (Thermo scientific, UK) spots were excised from the gel
47
48
and washed in 50mM ammonium bicarbonate pH 8.0 in 50% acetonitrile (VWR International) to a
49
50
51 complete destaining. The gel pieces were re-suspended in 50mM ammonium bicarbonate pH 8.0
52
53 containing 100 ng of trypsin (Sigma-Aldrich) and incubated for 2 hr at 4°C and overnight at 37°C.
54
55
The supernatant containing the resulting peptide mixtures was removed and the gel pieces were re-
56
57
58 extracted with acetonitrile. The two fractions were then collected and freeze-dried.
59
60

61
62
63
8
64 Page 8 of 47
65