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1
Optimizing the measurement of mitochondrial protein synthesis in human
2
skeletal muscle
3 4
Nicholas A. Burd1, Nicolas Tardif3, Olav Rooyackers2,3 , and Luc J.C. van Loon4*
5 6
1
7
Urbana, Illinois, United States, 2Department of Anaesthesia and Intensive Care, Karolinska
8
University Hospital, Huddinge, Sweden, 3Department of Clinical Science, Intervention and
9
Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden, 4Maastricht University,
10
Department of Kinesiology and Community Health, University of Illinois at Urbana-Champaign,
Maastricht, the Netherlands
11 12
Running title:
Mitochondrial protein synthesis in humans
13
Key words:
stable isotope tracers, exercise, nutrition, critically ill, muscle mass
14 15
Address for correspondence
16
Luc J.C. van Loon, PhD
17
Department of Human Movement Sciences
18
Maastricht University Medical Centre+
19
PO Box 616
20
6200 MD Maastricht
21
The Netherlands
22
E-mail: [email protected]
23
Phone: +31 43 3881397
24
Fax:
+31 43 3670976
25
1
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26
Abstract
27
The measurement of mitochondrial protein synthesis after food ingestion, contractile activity,
28
and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the
29
longer-term. Studies have shown that protein ingestion stimulates mitochondrial protein
30
synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial
31
protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be
32
no measurable differences in mitochondrial protein synthesis between chronic-ill patients and
33
aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a
34
more advanced age. In this paper, we discuss the challenges involved in the measurement of
35
human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid
36
tracer methods. Practical guidelines are discussed to improve the reliability of the measurement
37
of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein
38
synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle
39
mass and performance outcomes in both the healthy and disease populations requires more work,
40
but we emphasize that the measurements need to be reliable to be of any value to the field.
41 42 43 44 45 46 47 48 49
2
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INTRODUCTION
51
Skeletal muscle proteins are constantly and simultaneously being synthesized and degraded.
52
Stable isotope labeled amino acids are commonly used as tracers to capture this dynamic process
53
of protein turnover. The measurement of specific muscle protein fractional synthesis rates (e.g.,
54
myofibrillar, sarcoplasmic, mitochondrial) may provide more detailed information towards the
55
skeletal muscle adaptive response when compared with the measurement of mixed muscle
56
protein synthesis rates. In the first in vivo measurements of mitochondrial protein synthesis rates
57
in human skeletal muscle tissue, it was observed that postabsorptive mitochondrial protein
58
synthesis rates are considerably lower in middle aged and older populations when compared with
59
younger adults (Rooyackers et al. 1996). The measurement of the synthesis rates of sarcoplasmic
60
proteins demonstrated no age-related differences (Balagopal et al. 1997). The reduced
61
mitochondrial protein synthesis rates with aging may be responsible for the decline in
62
mitochondrial protein content and aerobic capacity (Short et al. 2005). Since this original report
63
(Rooyackers et al. 1996), scientists have measured mitochondrial protein synthesis rates after
64
food intake, during and after exercise, and various disease states for more qualitative insight into
65
the mitochondrial adaptations that may arise in the longer-term (Table 1-3).
66
In this paper we discuss the difficulties involved with the in vivo measurement of skeletal muscle
67
mitochondrial proteins synthesis rates. We have provided recommendations to minimize the
68
problems that may arise during the isolation and subsequent analysis of mitochondrial protein
69
synthesis. Also, we briefly discuss the potential consequence(s) of the observed changes in
70
mitochondrial protein synthesis rates after food ingestion, exercise, and disease for
71
understanding and modulating the skeletal muscle adaptation. The accurate measurement of the
72
mitochondrial protein synthetic response under the variety of conditions may provide detailed 3
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information for the prescription of more effective strategies to maintain (or enhance) muscle
74
health and performance in both the healthy and chronic diseased populations.
75 76
THE MEASUREMENT OF MITOCHONDRIAL PROTEIN SYNTHESIS
77
The in vivo measurement of the fractional synthesis rates (FSR) of mitochondrial proteins is
78
possible by using a prime-constant infusion or large dose injection of stable isotope amino acid
79
tracers into the blood stream which is subsequently coupled with frequent blood sampling and
80
collection of skeletal muscle biopsies (Rennie et al. 1994). Certainly some interest has recently
81
been sparked with the use of heavy water methods to characterize the mitochondrial protein
82
synthetic response over a period of days to weeks, but this will not be the focus of this paper. For
83
more established methods, the calculation of mitochondrial protein FSR requires the
84
quantification of the amount of isotope labeled-amino acid (tracer) that is incorporated into
85
skeletal muscle mitochondrial protein against its naturally abundant unlabeled amino acid
86
(tracee). To achieve this, the mitochondrial protein fractions must be isolated from the tissue of
87
interest as ‘purely’ as possible. The change in mitochondrial protein labeling is measured
88
between the two skeletal muscle biopsies collected over time in relation to the tracer labeling in
89
the precursor pool (muscle aminoacyl-tRNA, muscle free amino acids, or the plasma free amino
90
acids). The FSR value is usually expressed in percent per hour or percent per day (Wolfe and
91
Chinkes 2005).
92
The mitochondria are unique from most other proteins in the cells. A subset of the mitochondrial
93
proteins are encoded and synthesized within the mitochondria themselves and thus the precursor
94
pool for their synthesis is also located within the mitochondria. However, most workers use the
95
intracellular (or extracellular) precursor pools for the enrichment value for the mitochondrial 4
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FSR calculations. This approach seems reliable as the majority of the mitochondria proteins are
97
encoded by the nucleus and synthesized by the cellular systems. Therefore the precursor pool
98
labeling within the mitochondrial should be very similar to the labeling in the surrogate precursor
99
pool (i.e., muscle intracellular free amino acids).
100
The measurement of the in vivo synthesis rates of mitochondrial proteins are not as straight
101
forward when compared to the measurement of the synthesis rates of other specific muscle
102
protein fractions. However, many of the recommendations provided in this paper will also be of
103
relevance to assess protein synthesis of mixed muscle and other specific muscle proteins
104
(myofibrillar or sarcoplasmic). Below, we will address the general tracer principles (e.g. tracer
105
selection, timing of measurements, and analytical techniques) as well as mitochondria specific
106
issues (e.g. isolation of mitochondria) associated with the measurement of mitochondrial protein
107
synthesis rates in vivo in skeletal muscle tissue.
108 109
Tracer selection
110
The selection of the amino acid tracer, or its labeling position, will always be dependent on the
111
type of mass spectrometry analysis and the finances available to the researcher. Prior to
112
participant recruitment, it is important to collect a detailed description of the participants’
113
involvement in prior stable isotope amino acid tracer experiments. The background of two (or
114
more) isotopomers of phenylalanine (e.g., [ring-2H5] or [15N]) in muscle protein with the
115
subsequent infusion of a third phenylalanine tracer (e.g., [ring-13C6]) may impair the precision
116
and accuracy of the mass spectrometry (Soop et al. 2012). To date, researchers have used a
117
variety of different stable isotope amino acid tracers for the measurement of mitochondrial
118
protein synthesis rates in humans. So far, multi-labeled phenylalanine or leucine tracers have 5
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been the preference amongst researchers (Table 1). These essential amino acids have reasonably
120
small pool sizes and no large transmembrane gradient into the different tissues ensuring that a
121
steady state will be reached in a relatively short time and with an affordable amount of tracers.
122
As discussed below, the specific amino acid tracer(s) selected for the stable isotope infusion
123
experiment influences the mitochondrial protein enrichment analysis.
124 125
Timing of measurement
126
A proper priming dose of stable isotope labeled amino acid tracer (phenylalanine: 2 µmol/kg or
127
leucine: 8 µmol/kg) allows for equilibration of the muscle free pool within ~1 h after the
128
initiation of the constant stable isotope amino acid infusion. However, care should be taken when
129
a patient group with significant changes in the amino acid pool size is studied. In these patients
130
an adjusted priming dose of tracer might be necessary.
131
It is often difficult to determine the ‘optimal’ time point to collect a skeletal muscle biopsy for
132
the measurement of sub-fractional muscle protein synthetic responses following an intervention
133
(e.g., food intake or exercise) that uses the primed constant infusion method. The goal of many
134
studies is to examine the responses in both the myofibrillar and mitochondrial protein pools to
135
improve the understanding of the skeletal muscle tissue adaptive response. Recent humans
136
studies show that the time course of maximal post-exercise responses may differ between
137
mitochondrial and myofibrillar protein synthesis rates during recovery from exercise (Burd et al.
138
2010; Breen et al. 2011; Burd et al. 2012; Di Donato et al. 2014). The stimulation of
139
mitochondrial protein synthesis is most robust at ~24 h of post-exercise recovery (Burd et al.
140
2012; Di Donato et al. 2014), whereas myofibrillar protein synthetic responses are maximal
141
during the more acute post-exercise recovery phase (0-6 h). As such, multiple muscle biopsies 6
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are likely required to investigate both exercise-induced myofibrillar and mitochondrial protein
143
synthetic responses simultaneously.
144
The length of tracer incorporation in the mitochondrial protein pool between muscle biopsy
145
sampling time points may also have important analytical implications for the assessment of
146
mitochondrial protein synthesis. Analytical difficulties may arise when attempting to measure the
147
change in labeling between two muscle biopsies collected over a short incorporation period (0-1
148
h). Such a short time interval only allows for small changes in muscle protein enrichments to
149
occur between muscle biopsies as most skeletal muscle proteins tend to have slow(er) protein
150
turnover rates when compared to tissue protein of other organs. The evidence suggests that the
151
use of multi-labeled amino acid tracers, as compared to single-labeled tracers, will improve the
152
analytical reproducibility of the measured muscle protein-bound enrichments after such relative
153
short incorporation periods due to the elimination of a large background enrichment occurring
154
from the natural abundance of stable isotopes (Yarasheski et al. 1992; Balagopal et al. 1996).
155
However, original work showed that a longer tracer incorporation period between muscle
156
biopsies, at least ≥5 h apart (Balagopal et al. 1996), in the primed constant infusion method may
157
be necessary to allow for adequate mitochondrial protein labeling and isotopic enrichment
158
measurements that are reproducible when using GC-C-IRMS analysis (Yarasheski, Smith et al.
159
1992; Balagopal, Ford et al. 1996). Although, the flood dose method can achieve this in a shorter
160
time frame due to the large amount of tracer and tracee amino acids that are intravenously
161
injected. Overall, we recommend that researchers attempt to achieve adequate mitochondrial
162
protein labeling between skeletal muscle biopsies during primed constant infusions as otherwise
163
the measurement of mitochondrial protein FSR, or any other protein fraction, will have no
164
significance due to its unreliability. 7
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Skeletal muscle biopsy sample amount
166
The Bergström needle muscle biopsy technique is regularly used to collect skeletal muscle tissue
167
in human intervention trials. The small relative contribution of mitochondrial proteins, as
168
compared to myofibrillar proteins, to overall skeletal muscle protein mass (estimates tend to
169
revolve ~10% (Balagopal et al. 1996)) usually necessitates that larger skeletal muscle biopsy
170
samples are collected to gain adequate amounts of mitochondrial protein for adequate signal on
171
the mass spectrometer. This notion is still true despite the fact that technical improvements in the
172
mass spectrometry analysis have decreased the amount of tissue material needed for other types
173
of analysis. The needle muscle biopsy technique only requires that a small population of muscle
174
fibers is sampled from the muscle of choice (commonly the vastus lateralis) for the
175
determination of mitochondrial protein synthesis.
176
population of muscle fibers is representative of the whole muscle. The needle muscle biopsy
177
technique does not allow for repeat sampling of the same pocket of muscle fibers with the same
178
fiber type composition with sequential muscle biopsies. However, the concern of sampling
179
different muscle fiber types with the needle muscle biopsy technique and the subsequent
180
influence on the calculated muscle protein synthetic response is minimal (Mittendorfer et al.
181
2005; Volpi et al. 2008).
182
Overall, a range of different amounts of tissue have been used to assess mitochondrial protein
183
synthesis rates in humans. In particular, usually
184
recommended for reliable measurements of mitochondrial protein synthesis rates in humans
185
using GC-MS or GC-C-IRMS analysis (Rooyackers et al. 1997). This amount allows for an
186
improved representation of mitochondria protein (~750-1000 µg mitochondrial enriched protein)
187
to be obtained during the extraction procedure, especially since the mitochondria in skeletal 8
As such, it is assumed that this small
>100 mg of skeletal muscle tissue is
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muscle tissue are not homogenously distributed. The choice of amino acid tracer can have a
189
direct influence on the amount of skeletal muscle tissue required for the mitochondrial protein
190
extraction procedure. The higher relative concentration of leucine (~9%), as compared to
191
phenylalanine (~4%), in skeletal muscle proteins (Reeds et al. 1994; Burd et al. 2013) will in
192
principle allow for a greater signal intensity on the mass spectrometers for the same absolute
193
amount of skeletal muscle tissue. However, signal intensity will only be of value when adequate
194
amounts of mitochondrial proteins are isolated during the extraction procedure.
195 196
Mitochondrial protein extraction procedure
197
Skeletal muscle mitochondria are often thought to exist in two distinct sub-populations, the
198
subsarcolemmal and intermyofibrillar mitochondria (Krieger et al. 1980). There is evidence,
199
however, that demonstrates the mitochondrial protein pools may exist in networks as opposed to
200
completely separate populations within human skeletal muscle tissue (Dahl et al. 2014). Thus,
201
more work is necessary to determine if the subsarolemmal and intermyofibrillar mitochondria are
202
interconnected or function independently. Based on the idea that the mitochondria may exist in
203
sub-populations within muscle, it has been shown that the mitochondrial sub-populations may
204
respond differently in terms of morphological changes and content to anabolic/catabolic stimuli
205
(Krieger, Tate et al. 1980; Chomentowski et al. 2011). The effect of endurance exercise on the
206
mitochondrial adaptive response does not seem to differ between the subsarcolemmal or
207
intermyofibrillar mitochondria in human skeletal muscle tissue (Hoppeler et al. 1973; Hoppeler
208
et al. 1985). However, more recent data has shown that exercise may preferentially stimulate
209
adaptations in intermyofibrillar mitochondria when compared to the subsarcolemmal
210
compartment in mouse skeletal muscle tissue (Picard et al. 2013). 9
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In the original report, Rooyackers et al (Rooyackers et al. 1996) measured only subsarcolemmal
212
mitochondrial protein synthesis rates to preserve the muscle for the calculation of myofibrillar
213
protein synthesis rates. To release the intermyofibrillar mitochondria from the myofibrils
214
requires the proteins to be incubated with proteolytic enzymes and ultimately this extraction
215
approach ‘interferes’ with the measurement of the myofibrillar protein enrichment (Rooyackers
216
et al. 1996). Wilkinson et al. (Wilkinson et al. 2008) avoided interfering with the myofibrillar
217
extraction procedure by using a strong mechanical homogenization procedure. This approach has
218
been reported to be effective in releasing the intermyofibrillar mitochondria from the myofibrils
219
(Bezaire et al. 2004). However, the researchers were not able to isolate sufficient amounts of
220
intermyofibrillar or subsarcolemmal mitochondria for the enrichment analysis and therefore both
221
skeletal muscle mitochondrial fractions were combined for the enrichment analysis (Wilkinson,
222
Phillips et al. 2008). It is important to note that eliminating the use of the proteolytic agents from
223
the extraction procedure will lower the yield of mitochondrial protein recovery during
224
fractionation by centrifugation (Frezza et al. 2007). This further underlines the importance of
225
using an adequate amount of muscle sample for the extraction procedure when the goal is to
226
determine both myofibrillar and mitochondrial protein synthesis.
227
The tissue homogenization hardware used for the mitochondrial protein extraction is typically a
228
Dounce glass or a Potter-Elvehjem homogenizer using a glass tube and a teflon pestle coupled
229
with >100 mg of wet skeletal muscle tissue. The tissue can be carefully minced with a pair of
230
scissors to improve the yield of the mitochondrial protein before it is homogenized. The whole
231
procedure should be performed at a low temperature (using cold buffers and centrifugation at
232
4˚C) to maintain the integrity of the mitochondria protein and to preserve the activities of the
233
mitochondrial enzymes. The mitochondrial extraction homogenization buffer contains 10
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ingredients to prevent the mitochondria from bursting and losing its matrix content (e.g.,
235
potassium chloride [KCL] or sucrose). Some type of metal ion chelator, usually EDTA, is
236
included to bind Mg2+ and Ca2+ to, expectantly, prevent activation of the proteolytic enzymes.
237
Antioxidants are included in the buffer to protect the proteins against oxidation and also
238
chemical compounds to stable the pH level of the buffer (e.g., Tris HCL) (Safdar et al. 2011).
239
Finally, phosphatase and protease inhibitors can be added to the buffer if a portion of the
240
sarcoplasmic fraction is used for Western blotting analysis (an example of a good extraction
241
buffer can be found in references (Safdar, Bourgeois et al. 2011; Burd et al. 2012) and a full
242
description of the procedure is provided in the supplemental file). For mitochondrial respiration
243
studies, bovine serum albumin (BSA) is commonly added to bind certain molecules and prevent
244
proteases from degrading the mitochondrial proteins. The presence of BSA will render the
245
homogenate useless for Western blotting analysis and/or the measurement of sarcoplasmic
246
protein synthesis rates. As a rule, no proteins (e.g., BSA) should be added to these procedures
247
since these proteins will be unlabeled and dilute the tracer in the protein of interest and give
248
falsely low synthesis rates.
249
A scheme of differential centrifugation is usually used to isolate the mitochondrial protein
250
enriched muscle protein fractions. Newer techniques are also available to isolate mitochondria
251
based on immune precipitation principle (Hornig-Do et al. 2009; Franko et al. 2013), but this
252
approach has not been validated for the measurement of mitochondrial protein FSR. Differential
253
centrifugation separates the muscle proteins suspended in the buffer based on their size and how
254
they respond to the centrifugal force. This approach does not produce a pure mitochondrial
255
protein fraction. The mitochondrial pellet will contain a contamination of contractile and other
256
sarcoplasmic proteins and thus should be considered a mitochondrial protein ‘enriched’ pellet 11
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(Jaleel et al. 2008). Ultra-centrifugation, due to the high-speed spin, may improve the separation
258
of these fractions (unpublished observations), but an ultracentrifuge is not always readily
259
available. The major objective is to minimize the cross contamination from the other major
260
protein fractions, especially the contractile proteins, with adequate speed of centrifugation and
261
precise pipetting during separation of the muscle protein fractions. In general, the larger protein
262
fraction, which includes the myofibrillar protein fraction, is first spun down at a low(er) speed
263
(~700×g). The obtained supernatant contains the subsarcolemma mitochondria and the
264
sarcoplasmic proteins. A second centrifugation of the supernatant at the same low speed results
265
in less contamination of the mitochondrial fraction with the myofibrillar proteins (unpublished
266
observation). Subsequently, the mitochondria are spun down from the supernatant by spinning at
267
a higher speed (~12000×g). The choice of the centrifugation speed should be based on a fine
268
balance between maximizing the amount of mitochondrial protein and maintenance of the level
269
of mitochondrial purity of the fraction. The faster the centrifugation speed the more
270
mitochondrial protein will be precipitated, but at the same time there is increased risk of
271
contamination by the presence of other muscle protein sub-fractions. The obtained mitochondrial
272
pellet is washed with higher ion strength buffer to remove part of the impurities and spun at the
273
same speed (~12000×g). A hydrolysis procedure is performed to free the amino acids from the
274
mitochondrial enriched protein pellet. The free amino acids are purified over an ion-exchange
275
resin to clean the obtained mitochondrial derived amino acids. Afterwards, the amino acids can
276
be converted to a specific derivative depending on the analytical method to be used.
277
It is worth taking into consideration that the mitochondrial extraction procedure does not extract
278
all skeletal muscle mitochondria. Rasmussen et al.(Rasmussen et al. 1997; Rasmussen et al.
279
2000) used a specialized equipment to achieve mitochondria extraction yields in the range of 40 12
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280
– 60% of total mitochondria. However, standard extraction procedures (as we have described)
281
will generally yield ~20% of total mitochondria. Assessment of the purity of the mitochondrial
282
protein enriched fraction is highly recommended for the mitochondrial protein synthesis
283
measurement. It has been reported that isolated mitochondrial protein enriched pellet has ~190%
284
(3-fold) greater citrate synthase activity when compared to the mixed muscle homogenate
285
(Wilkinson et al. 2008). The purity of the mitochondrial enriched compartment can also be tested
286
by two other methods. The first method is the measurement of the non-mitochondrial ATPase
287
activities in the presence or absence of oligomycin (an inhibitor of the mitochondrial ATPase
288
complex). The differential centrifugation method has been demonstrated to result in ~5-10%
289
remaining ATPase activity in the presence of oligomycin with the contamination stemming from
290
the sarcolemma, myofibrils, or endoplasmic reticulum (Krieger et al. 1980; Cogswell et al. 1993;
291
Rooyackers et al. 1996). The second method is the quantification of the contaminant by the
292
measurement of the expression of proteins specific to the other intracellular compartments
293
(nucleus, cytoplasm, endoplasmic reticulum, and myofibrils in the mitochondrial protein fraction
294
(Hornig-Do et al. 2009). In our opinion, the second method should be used to monitor the
295
presence of contaminants in the mitochondria-enriched pellet. The mitochondrial extraction
296
protocol should be adapted in order to obtain the purest mitochondrial protein fraction possible.
297 298
Mitochondrial protein enrichment analysis
299
The original work of Rooyackers et al. (Rooyackers et al. 1996) used gas chromatograph-
300
combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the analysis of mitochondrial
301
protein bound enrichments. The advantage with GC-C-IRMS analysis is the greater precision
302
(~6×) (Yarasheski et al. 1992; Balagopal et al. 1996; Rooyackers et al. 1997) for the accurate 13
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detection of very low enrichments in the mitochondrial protein-bound samples than with gas
304
chromatography mass spectrometry (GC-MS) analysis (Calder et al. 1992; Slater et al. 1995;
305
Patterson et al. 1997). To allow for separation of the amino acids by gas chromatography, it is
306
necessary to increase the volatility by chemical derivatization (Shinebarger et al. 2002). For
307
carbon-labeled amino acid tracers, the additional carbons that are eventually added during the
308
derivatization process suggests that when applying single-labeled tracers a longer incorporation
309
time is warranted to minimize the dilution of the
310
molecule and, as such, increase the precision of the enrichment measurement. (Yarasheski et al.
311
1992; Balagopal et al. 1996). The tracer incorporation length can likely be shortened when
312
applying tracers with two or more labels (e.g., 3 h of incorporation time between two consecutive
313
muscle biopsies). Previous work has demonstrated that the coefficients of variation in using the
314
GC-C-IRMS for the analysis of the mitochondrial protein-bound 1-[13C]leucine enrichments
315
were 0.027% from measurements of 5 separate pieces of the same pig muscle (Rooyackers et al.
316
1997). This reproducibility is only possible provided adequate amounts of skeletal muscle tissue
317
are used for the extraction procedure (>100 mg) and the tracer incorporation time, regardless of
318
the tracer applied, is long enough to allow for sufficient labeling of the mitochondrial protein
319
pool.
320
It is a common misconception that the greater precision (
Page 1 of 32
1
Optimizing the measurement of mitochondrial protein synthesis in human
2
skeletal muscle
3 4
Nicholas A. Burd1, Nicolas Tardif3, Olav Rooyackers2,3 , and Luc J.C. van Loon4*
5 6
1
7
Urbana, Illinois, United States, 2Department of Anaesthesia and Intensive Care, Karolinska
8
University Hospital, Huddinge, Sweden, 3Department of Clinical Science, Intervention and
9
Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden, 4Maastricht University,
10
Department of Kinesiology and Community Health, University of Illinois at Urbana-Champaign,
Maastricht, the Netherlands
11 12
Running title:
Mitochondrial protein synthesis in humans
13
Key words:
stable isotope tracers, exercise, nutrition, critically ill, muscle mass
14 15
Address for correspondence
16
Luc J.C. van Loon, PhD
17
Department of Human Movement Sciences
18
Maastricht University Medical Centre+
19
PO Box 616
20
6200 MD Maastricht
21
The Netherlands
22
E-mail: [email protected]
23
Phone: +31 43 3881397
24
Fax:
+31 43 3670976
25
1
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Page 2 of 32
26
Abstract
27
The measurement of mitochondrial protein synthesis after food ingestion, contractile activity,
28
and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the
29
longer-term. Studies have shown that protein ingestion stimulates mitochondrial protein
30
synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial
31
protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be
32
no measurable differences in mitochondrial protein synthesis between chronic-ill patients and
33
aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a
34
more advanced age. In this paper, we discuss the challenges involved in the measurement of
35
human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid
36
tracer methods. Practical guidelines are discussed to improve the reliability of the measurement
37
of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein
38
synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle
39
mass and performance outcomes in both the healthy and disease populations requires more work,
40
but we emphasize that the measurements need to be reliable to be of any value to the field.
41 42 43 44 45 46 47 48 49
2
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50
INTRODUCTION
51
Skeletal muscle proteins are constantly and simultaneously being synthesized and degraded.
52
Stable isotope labeled amino acids are commonly used as tracers to capture this dynamic process
53
of protein turnover. The measurement of specific muscle protein fractional synthesis rates (e.g.,
54
myofibrillar, sarcoplasmic, mitochondrial) may provide more detailed information towards the
55
skeletal muscle adaptive response when compared with the measurement of mixed muscle
56
protein synthesis rates. In the first in vivo measurements of mitochondrial protein synthesis rates
57
in human skeletal muscle tissue, it was observed that postabsorptive mitochondrial protein
58
synthesis rates are considerably lower in middle aged and older populations when compared with
59
younger adults (Rooyackers et al. 1996). The measurement of the synthesis rates of sarcoplasmic
60
proteins demonstrated no age-related differences (Balagopal et al. 1997). The reduced
61
mitochondrial protein synthesis rates with aging may be responsible for the decline in
62
mitochondrial protein content and aerobic capacity (Short et al. 2005). Since this original report
63
(Rooyackers et al. 1996), scientists have measured mitochondrial protein synthesis rates after
64
food intake, during and after exercise, and various disease states for more qualitative insight into
65
the mitochondrial adaptations that may arise in the longer-term (Table 1-3).
66
In this paper we discuss the difficulties involved with the in vivo measurement of skeletal muscle
67
mitochondrial proteins synthesis rates. We have provided recommendations to minimize the
68
problems that may arise during the isolation and subsequent analysis of mitochondrial protein
69
synthesis. Also, we briefly discuss the potential consequence(s) of the observed changes in
70
mitochondrial protein synthesis rates after food ingestion, exercise, and disease for
71
understanding and modulating the skeletal muscle adaptation. The accurate measurement of the
72
mitochondrial protein synthetic response under the variety of conditions may provide detailed 3
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information for the prescription of more effective strategies to maintain (or enhance) muscle
74
health and performance in both the healthy and chronic diseased populations.
75 76
THE MEASUREMENT OF MITOCHONDRIAL PROTEIN SYNTHESIS
77
The in vivo measurement of the fractional synthesis rates (FSR) of mitochondrial proteins is
78
possible by using a prime-constant infusion or large dose injection of stable isotope amino acid
79
tracers into the blood stream which is subsequently coupled with frequent blood sampling and
80
collection of skeletal muscle biopsies (Rennie et al. 1994). Certainly some interest has recently
81
been sparked with the use of heavy water methods to characterize the mitochondrial protein
82
synthetic response over a period of days to weeks, but this will not be the focus of this paper. For
83
more established methods, the calculation of mitochondrial protein FSR requires the
84
quantification of the amount of isotope labeled-amino acid (tracer) that is incorporated into
85
skeletal muscle mitochondrial protein against its naturally abundant unlabeled amino acid
86
(tracee). To achieve this, the mitochondrial protein fractions must be isolated from the tissue of
87
interest as ‘purely’ as possible. The change in mitochondrial protein labeling is measured
88
between the two skeletal muscle biopsies collected over time in relation to the tracer labeling in
89
the precursor pool (muscle aminoacyl-tRNA, muscle free amino acids, or the plasma free amino
90
acids). The FSR value is usually expressed in percent per hour or percent per day (Wolfe and
91
Chinkes 2005).
92
The mitochondria are unique from most other proteins in the cells. A subset of the mitochondrial
93
proteins are encoded and synthesized within the mitochondria themselves and thus the precursor
94
pool for their synthesis is also located within the mitochondria. However, most workers use the
95
intracellular (or extracellular) precursor pools for the enrichment value for the mitochondrial 4
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FSR calculations. This approach seems reliable as the majority of the mitochondria proteins are
97
encoded by the nucleus and synthesized by the cellular systems. Therefore the precursor pool
98
labeling within the mitochondrial should be very similar to the labeling in the surrogate precursor
99
pool (i.e., muscle intracellular free amino acids).
100
The measurement of the in vivo synthesis rates of mitochondrial proteins are not as straight
101
forward when compared to the measurement of the synthesis rates of other specific muscle
102
protein fractions. However, many of the recommendations provided in this paper will also be of
103
relevance to assess protein synthesis of mixed muscle and other specific muscle proteins
104
(myofibrillar or sarcoplasmic). Below, we will address the general tracer principles (e.g. tracer
105
selection, timing of measurements, and analytical techniques) as well as mitochondria specific
106
issues (e.g. isolation of mitochondria) associated with the measurement of mitochondrial protein
107
synthesis rates in vivo in skeletal muscle tissue.
108 109
Tracer selection
110
The selection of the amino acid tracer, or its labeling position, will always be dependent on the
111
type of mass spectrometry analysis and the finances available to the researcher. Prior to
112
participant recruitment, it is important to collect a detailed description of the participants’
113
involvement in prior stable isotope amino acid tracer experiments. The background of two (or
114
more) isotopomers of phenylalanine (e.g., [ring-2H5] or [15N]) in muscle protein with the
115
subsequent infusion of a third phenylalanine tracer (e.g., [ring-13C6]) may impair the precision
116
and accuracy of the mass spectrometry (Soop et al. 2012). To date, researchers have used a
117
variety of different stable isotope amino acid tracers for the measurement of mitochondrial
118
protein synthesis rates in humans. So far, multi-labeled phenylalanine or leucine tracers have 5
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been the preference amongst researchers (Table 1). These essential amino acids have reasonably
120
small pool sizes and no large transmembrane gradient into the different tissues ensuring that a
121
steady state will be reached in a relatively short time and with an affordable amount of tracers.
122
As discussed below, the specific amino acid tracer(s) selected for the stable isotope infusion
123
experiment influences the mitochondrial protein enrichment analysis.
124 125
Timing of measurement
126
A proper priming dose of stable isotope labeled amino acid tracer (phenylalanine: 2 µmol/kg or
127
leucine: 8 µmol/kg) allows for equilibration of the muscle free pool within ~1 h after the
128
initiation of the constant stable isotope amino acid infusion. However, care should be taken when
129
a patient group with significant changes in the amino acid pool size is studied. In these patients
130
an adjusted priming dose of tracer might be necessary.
131
It is often difficult to determine the ‘optimal’ time point to collect a skeletal muscle biopsy for
132
the measurement of sub-fractional muscle protein synthetic responses following an intervention
133
(e.g., food intake or exercise) that uses the primed constant infusion method. The goal of many
134
studies is to examine the responses in both the myofibrillar and mitochondrial protein pools to
135
improve the understanding of the skeletal muscle tissue adaptive response. Recent humans
136
studies show that the time course of maximal post-exercise responses may differ between
137
mitochondrial and myofibrillar protein synthesis rates during recovery from exercise (Burd et al.
138
2010; Breen et al. 2011; Burd et al. 2012; Di Donato et al. 2014). The stimulation of
139
mitochondrial protein synthesis is most robust at ~24 h of post-exercise recovery (Burd et al.
140
2012; Di Donato et al. 2014), whereas myofibrillar protein synthetic responses are maximal
141
during the more acute post-exercise recovery phase (0-6 h). As such, multiple muscle biopsies 6
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are likely required to investigate both exercise-induced myofibrillar and mitochondrial protein
143
synthetic responses simultaneously.
144
The length of tracer incorporation in the mitochondrial protein pool between muscle biopsy
145
sampling time points may also have important analytical implications for the assessment of
146
mitochondrial protein synthesis. Analytical difficulties may arise when attempting to measure the
147
change in labeling between two muscle biopsies collected over a short incorporation period (0-1
148
h). Such a short time interval only allows for small changes in muscle protein enrichments to
149
occur between muscle biopsies as most skeletal muscle proteins tend to have slow(er) protein
150
turnover rates when compared to tissue protein of other organs. The evidence suggests that the
151
use of multi-labeled amino acid tracers, as compared to single-labeled tracers, will improve the
152
analytical reproducibility of the measured muscle protein-bound enrichments after such relative
153
short incorporation periods due to the elimination of a large background enrichment occurring
154
from the natural abundance of stable isotopes (Yarasheski et al. 1992; Balagopal et al. 1996).
155
However, original work showed that a longer tracer incorporation period between muscle
156
biopsies, at least ≥5 h apart (Balagopal et al. 1996), in the primed constant infusion method may
157
be necessary to allow for adequate mitochondrial protein labeling and isotopic enrichment
158
measurements that are reproducible when using GC-C-IRMS analysis (Yarasheski, Smith et al.
159
1992; Balagopal, Ford et al. 1996). Although, the flood dose method can achieve this in a shorter
160
time frame due to the large amount of tracer and tracee amino acids that are intravenously
161
injected. Overall, we recommend that researchers attempt to achieve adequate mitochondrial
162
protein labeling between skeletal muscle biopsies during primed constant infusions as otherwise
163
the measurement of mitochondrial protein FSR, or any other protein fraction, will have no
164
significance due to its unreliability. 7
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165
Skeletal muscle biopsy sample amount
166
The Bergström needle muscle biopsy technique is regularly used to collect skeletal muscle tissue
167
in human intervention trials. The small relative contribution of mitochondrial proteins, as
168
compared to myofibrillar proteins, to overall skeletal muscle protein mass (estimates tend to
169
revolve ~10% (Balagopal et al. 1996)) usually necessitates that larger skeletal muscle biopsy
170
samples are collected to gain adequate amounts of mitochondrial protein for adequate signal on
171
the mass spectrometer. This notion is still true despite the fact that technical improvements in the
172
mass spectrometry analysis have decreased the amount of tissue material needed for other types
173
of analysis. The needle muscle biopsy technique only requires that a small population of muscle
174
fibers is sampled from the muscle of choice (commonly the vastus lateralis) for the
175
determination of mitochondrial protein synthesis.
176
population of muscle fibers is representative of the whole muscle. The needle muscle biopsy
177
technique does not allow for repeat sampling of the same pocket of muscle fibers with the same
178
fiber type composition with sequential muscle biopsies. However, the concern of sampling
179
different muscle fiber types with the needle muscle biopsy technique and the subsequent
180
influence on the calculated muscle protein synthetic response is minimal (Mittendorfer et al.
181
2005; Volpi et al. 2008).
182
Overall, a range of different amounts of tissue have been used to assess mitochondrial protein
183
synthesis rates in humans. In particular, usually
184
recommended for reliable measurements of mitochondrial protein synthesis rates in humans
185
using GC-MS or GC-C-IRMS analysis (Rooyackers et al. 1997). This amount allows for an
186
improved representation of mitochondria protein (~750-1000 µg mitochondrial enriched protein)
187
to be obtained during the extraction procedure, especially since the mitochondria in skeletal 8
As such, it is assumed that this small
>100 mg of skeletal muscle tissue is
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muscle tissue are not homogenously distributed. The choice of amino acid tracer can have a
189
direct influence on the amount of skeletal muscle tissue required for the mitochondrial protein
190
extraction procedure. The higher relative concentration of leucine (~9%), as compared to
191
phenylalanine (~4%), in skeletal muscle proteins (Reeds et al. 1994; Burd et al. 2013) will in
192
principle allow for a greater signal intensity on the mass spectrometers for the same absolute
193
amount of skeletal muscle tissue. However, signal intensity will only be of value when adequate
194
amounts of mitochondrial proteins are isolated during the extraction procedure.
195 196
Mitochondrial protein extraction procedure
197
Skeletal muscle mitochondria are often thought to exist in two distinct sub-populations, the
198
subsarcolemmal and intermyofibrillar mitochondria (Krieger et al. 1980). There is evidence,
199
however, that demonstrates the mitochondrial protein pools may exist in networks as opposed to
200
completely separate populations within human skeletal muscle tissue (Dahl et al. 2014). Thus,
201
more work is necessary to determine if the subsarolemmal and intermyofibrillar mitochondria are
202
interconnected or function independently. Based on the idea that the mitochondria may exist in
203
sub-populations within muscle, it has been shown that the mitochondrial sub-populations may
204
respond differently in terms of morphological changes and content to anabolic/catabolic stimuli
205
(Krieger, Tate et al. 1980; Chomentowski et al. 2011). The effect of endurance exercise on the
206
mitochondrial adaptive response does not seem to differ between the subsarcolemmal or
207
intermyofibrillar mitochondria in human skeletal muscle tissue (Hoppeler et al. 1973; Hoppeler
208
et al. 1985). However, more recent data has shown that exercise may preferentially stimulate
209
adaptations in intermyofibrillar mitochondria when compared to the subsarcolemmal
210
compartment in mouse skeletal muscle tissue (Picard et al. 2013). 9
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In the original report, Rooyackers et al (Rooyackers et al. 1996) measured only subsarcolemmal
212
mitochondrial protein synthesis rates to preserve the muscle for the calculation of myofibrillar
213
protein synthesis rates. To release the intermyofibrillar mitochondria from the myofibrils
214
requires the proteins to be incubated with proteolytic enzymes and ultimately this extraction
215
approach ‘interferes’ with the measurement of the myofibrillar protein enrichment (Rooyackers
216
et al. 1996). Wilkinson et al. (Wilkinson et al. 2008) avoided interfering with the myofibrillar
217
extraction procedure by using a strong mechanical homogenization procedure. This approach has
218
been reported to be effective in releasing the intermyofibrillar mitochondria from the myofibrils
219
(Bezaire et al. 2004). However, the researchers were not able to isolate sufficient amounts of
220
intermyofibrillar or subsarcolemmal mitochondria for the enrichment analysis and therefore both
221
skeletal muscle mitochondrial fractions were combined for the enrichment analysis (Wilkinson,
222
Phillips et al. 2008). It is important to note that eliminating the use of the proteolytic agents from
223
the extraction procedure will lower the yield of mitochondrial protein recovery during
224
fractionation by centrifugation (Frezza et al. 2007). This further underlines the importance of
225
using an adequate amount of muscle sample for the extraction procedure when the goal is to
226
determine both myofibrillar and mitochondrial protein synthesis.
227
The tissue homogenization hardware used for the mitochondrial protein extraction is typically a
228
Dounce glass or a Potter-Elvehjem homogenizer using a glass tube and a teflon pestle coupled
229
with >100 mg of wet skeletal muscle tissue. The tissue can be carefully minced with a pair of
230
scissors to improve the yield of the mitochondrial protein before it is homogenized. The whole
231
procedure should be performed at a low temperature (using cold buffers and centrifugation at
232
4˚C) to maintain the integrity of the mitochondria protein and to preserve the activities of the
233
mitochondrial enzymes. The mitochondrial extraction homogenization buffer contains 10
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ingredients to prevent the mitochondria from bursting and losing its matrix content (e.g.,
235
potassium chloride [KCL] or sucrose). Some type of metal ion chelator, usually EDTA, is
236
included to bind Mg2+ and Ca2+ to, expectantly, prevent activation of the proteolytic enzymes.
237
Antioxidants are included in the buffer to protect the proteins against oxidation and also
238
chemical compounds to stable the pH level of the buffer (e.g., Tris HCL) (Safdar et al. 2011).
239
Finally, phosphatase and protease inhibitors can be added to the buffer if a portion of the
240
sarcoplasmic fraction is used for Western blotting analysis (an example of a good extraction
241
buffer can be found in references (Safdar, Bourgeois et al. 2011; Burd et al. 2012) and a full
242
description of the procedure is provided in the supplemental file). For mitochondrial respiration
243
studies, bovine serum albumin (BSA) is commonly added to bind certain molecules and prevent
244
proteases from degrading the mitochondrial proteins. The presence of BSA will render the
245
homogenate useless for Western blotting analysis and/or the measurement of sarcoplasmic
246
protein synthesis rates. As a rule, no proteins (e.g., BSA) should be added to these procedures
247
since these proteins will be unlabeled and dilute the tracer in the protein of interest and give
248
falsely low synthesis rates.
249
A scheme of differential centrifugation is usually used to isolate the mitochondrial protein
250
enriched muscle protein fractions. Newer techniques are also available to isolate mitochondria
251
based on immune precipitation principle (Hornig-Do et al. 2009; Franko et al. 2013), but this
252
approach has not been validated for the measurement of mitochondrial protein FSR. Differential
253
centrifugation separates the muscle proteins suspended in the buffer based on their size and how
254
they respond to the centrifugal force. This approach does not produce a pure mitochondrial
255
protein fraction. The mitochondrial pellet will contain a contamination of contractile and other
256
sarcoplasmic proteins and thus should be considered a mitochondrial protein ‘enriched’ pellet 11
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(Jaleel et al. 2008). Ultra-centrifugation, due to the high-speed spin, may improve the separation
258
of these fractions (unpublished observations), but an ultracentrifuge is not always readily
259
available. The major objective is to minimize the cross contamination from the other major
260
protein fractions, especially the contractile proteins, with adequate speed of centrifugation and
261
precise pipetting during separation of the muscle protein fractions. In general, the larger protein
262
fraction, which includes the myofibrillar protein fraction, is first spun down at a low(er) speed
263
(~700×g). The obtained supernatant contains the subsarcolemma mitochondria and the
264
sarcoplasmic proteins. A second centrifugation of the supernatant at the same low speed results
265
in less contamination of the mitochondrial fraction with the myofibrillar proteins (unpublished
266
observation). Subsequently, the mitochondria are spun down from the supernatant by spinning at
267
a higher speed (~12000×g). The choice of the centrifugation speed should be based on a fine
268
balance between maximizing the amount of mitochondrial protein and maintenance of the level
269
of mitochondrial purity of the fraction. The faster the centrifugation speed the more
270
mitochondrial protein will be precipitated, but at the same time there is increased risk of
271
contamination by the presence of other muscle protein sub-fractions. The obtained mitochondrial
272
pellet is washed with higher ion strength buffer to remove part of the impurities and spun at the
273
same speed (~12000×g). A hydrolysis procedure is performed to free the amino acids from the
274
mitochondrial enriched protein pellet. The free amino acids are purified over an ion-exchange
275
resin to clean the obtained mitochondrial derived amino acids. Afterwards, the amino acids can
276
be converted to a specific derivative depending on the analytical method to be used.
277
It is worth taking into consideration that the mitochondrial extraction procedure does not extract
278
all skeletal muscle mitochondria. Rasmussen et al.(Rasmussen et al. 1997; Rasmussen et al.
279
2000) used a specialized equipment to achieve mitochondria extraction yields in the range of 40 12
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– 60% of total mitochondria. However, standard extraction procedures (as we have described)
281
will generally yield ~20% of total mitochondria. Assessment of the purity of the mitochondrial
282
protein enriched fraction is highly recommended for the mitochondrial protein synthesis
283
measurement. It has been reported that isolated mitochondrial protein enriched pellet has ~190%
284
(3-fold) greater citrate synthase activity when compared to the mixed muscle homogenate
285
(Wilkinson et al. 2008). The purity of the mitochondrial enriched compartment can also be tested
286
by two other methods. The first method is the measurement of the non-mitochondrial ATPase
287
activities in the presence or absence of oligomycin (an inhibitor of the mitochondrial ATPase
288
complex). The differential centrifugation method has been demonstrated to result in ~5-10%
289
remaining ATPase activity in the presence of oligomycin with the contamination stemming from
290
the sarcolemma, myofibrils, or endoplasmic reticulum (Krieger et al. 1980; Cogswell et al. 1993;
291
Rooyackers et al. 1996). The second method is the quantification of the contaminant by the
292
measurement of the expression of proteins specific to the other intracellular compartments
293
(nucleus, cytoplasm, endoplasmic reticulum, and myofibrils in the mitochondrial protein fraction
294
(Hornig-Do et al. 2009). In our opinion, the second method should be used to monitor the
295
presence of contaminants in the mitochondria-enriched pellet. The mitochondrial extraction
296
protocol should be adapted in order to obtain the purest mitochondrial protein fraction possible.
297 298
Mitochondrial protein enrichment analysis
299
The original work of Rooyackers et al. (Rooyackers et al. 1996) used gas chromatograph-
300
combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the analysis of mitochondrial
301
protein bound enrichments. The advantage with GC-C-IRMS analysis is the greater precision
302
(~6×) (Yarasheski et al. 1992; Balagopal et al. 1996; Rooyackers et al. 1997) for the accurate 13
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detection of very low enrichments in the mitochondrial protein-bound samples than with gas
304
chromatography mass spectrometry (GC-MS) analysis (Calder et al. 1992; Slater et al. 1995;
305
Patterson et al. 1997). To allow for separation of the amino acids by gas chromatography, it is
306
necessary to increase the volatility by chemical derivatization (Shinebarger et al. 2002). For
307
carbon-labeled amino acid tracers, the additional carbons that are eventually added during the
308
derivatization process suggests that when applying single-labeled tracers a longer incorporation
309
time is warranted to minimize the dilution of the
310
molecule and, as such, increase the precision of the enrichment measurement. (Yarasheski et al.
311
1992; Balagopal et al. 1996). The tracer incorporation length can likely be shortened when
312
applying tracers with two or more labels (e.g., 3 h of incorporation time between two consecutive
313
muscle biopsies). Previous work has demonstrated that the coefficients of variation in using the
314
GC-C-IRMS for the analysis of the mitochondrial protein-bound 1-[13C]leucine enrichments
315
were 0.027% from measurements of 5 separate pieces of the same pig muscle (Rooyackers et al.
316
1997). This reproducibility is only possible provided adequate amounts of skeletal muscle tissue
317
are used for the extraction procedure (>100 mg) and the tracer incorporation time, regardless of
318
the tracer applied, is long enough to allow for sufficient labeling of the mitochondrial protein
319
pool.
320
It is a common misconception that the greater precision (
