Mitochondrial function in skeletal muscle of patients with protracted critical illness and ICU-acquired weakness

Abstract

Background:
Mitochondrial damage occurs in the acute phase of critical illness, followed by activation of mitochondrial biogenesis in survivors. It has been hypothesized that bioenergetics failure of skeletal muscle may contribute to the development of ICU-acquired weakness. The aim of the present study was to determine whether mitochondrial dysfunction persists until protracted phase of critical illness.

Methods:
In this single-centre controlled-cohort ex vivo proof-of-concept pilot study, we obtained vastus lateralis biopsies from ventilated patients with ICU-acquired weakness (n = 8) and from age and sex-matched metabolically healthy controls (n = 8). Mitochondrial functional indices were measured in cytosolic context by high-resolution respirometry in tissue homogenates, activities of respiratory complexes by spectrophotometry and individual functional capacities were correlated with concentrations of electron transport chain key subunits from respiratory complexes II, III, IV and V measured by western blot.

Results:
The ability of aerobic ATP synthesis (OXPHOS) was reduced to ~54 % in ICU patients (p<0.01), in correlation with the depletion of complexes III (~38 % of control, p = 0.02) and IV (~26 % of controls, p<0.01) and without signs of mitochondrial uncoupling. When mitochondrial functional indices were adjusted to citrate synthase activity, OXPHOS and the activity of complexes I and IV were not different, whilst the activities of complexes II and III were increased in ICU patients 3-fold (p<0.01) respectively 2-fold (p<0.01).

Conclusions:
Compared to healthy controls, in ICU patients we have demonstrated a ~50 % reduction of the ability of skeletal muscle to synthetize ATP in mitochondria. We found a depletion of complex III and IV concentrations and relative increases in functional capacities of complex II and glycerol-3-phosphate dehydrogenase/complex III.

Full text

R E S E A R C H Open Access
Mitochondrial function in skeletal muscle of
patients with protracted critical illness and
ICU-acquired weakness
Kateřina Jiroutková
1*
, Adéla Krajčová
1,2
, Jakub Ziak
1
, Michal Fric
4
, Petr Waldauf
4
, Valér Džupa
3
, Jan Gojda
2
,
Vlasta Němcova-Fürstová
5
, Jan Kovář
5
, Moustafa Elkalaf
1
, Jan Trnka
1
and František Duška
1,6
Abstract
Background: Mitochondrial damage occurs in the acute phase of critical illness, followed by activation of mitochondrial
biogenesis in survivors. It has been hypothesized that bioenergetics failure of skeletal muscle may contribute to the
development of ICU-acquired weakness. The aim of the present study was to determine whether mitochondrial
dysfunction persists until protracted phase of critical illness.
Methods: In this single-centre controlled-cohort ex vivo proof-of-concept pilot study, we obtained vastus
lateralis biopsies from ventilated patients with ICU-acquired weakness (n = 8) and from age and sex-matched
metabolically healthy controls (n = 8). Mitochondrial functional indices were measured in cytosolic context by
high-resolution respirometry in tissue homogenates, activities of respiratory complexes by spectrophotometry
and individual functional capacities were correlated with concentrations of electron transport chain key subunits
from respiratory complexes II, III, IV and V measured by western blot.
Results: The ability of aerobic ATP synthesis (OXPHOS) was reduced to ~54 % in ICU patients (p<0.01), in correlation
with the depletion of complexes III (~38 % of control, p= 0.02) and IV (~26 % of controls, p<0.01) and without signs of
mitochondrial uncoupling. When mitochondrial functional indices were adjusted to citrate synthase activity,
OXPHOS and the activity of complexes I and IV were not different, whilst the activities of complexes II and III
were increased in ICU patients 3-fold (p<0.01) respectively 2-fold (p<0.01).
Conclusions: Compared to healthy controls, in ICU patients we have demonstrated a ~50 % reduction of the
ability of skeletal muscle to synthetize ATP in mitochondria. We found a depletion of complex III and IV concentrations
and relative increases in functional capacities of complex II and glycerol-3-phosphate dehydrogenase/complex III.
Background
Generalized inflammation and multi-organ failure in the
acute phase of critical illness are accompanied by
impairment of mitochondrial morphology [1] and func-
tion of skeletal muscle [2–5] and other organs [6, 7].
The extent of mitochondrial functional impairment cor-
relates with disease severity, intracellular ATP depletion
and outcomes [2]. It appears that the inability to meet
cellular ATP demand is caused by a global depletion of
functional mitochondria, as the reduction of respiratory
complex content [4] or activities [3] is proportional to
the reduction of citrate synthase activity (a measure of
mitochondrial content) with the exception of septic non-
survivors, in whom a disproportional reduction of
complex I activity has been demonstrated [2]. Moreover,
the ability to replenish functional mitochondria is an
independent predictor of survival of critical illness [4].
Little is known about mitochondrial function in
patients who do survive the acute phase of disease, but
fail to wean from mechanical ventilation and enter a
protracted phase of critical illness. We hypothesized that
bioenergetics failure would be present in the skeletal
muscle of patients with weaning failure and ICU-
acquired weakness as a result of mitochondrial uncoup-
ling and/or depletion. We performed muscle biopsies in
such patients, measured concentrations and activities of
* Correspondence: katka.nova@post.cz
1
Laboratory of Bioenergetics, Third Faculty of Medicine, Charles University in
Prague, Ruská 87, Prague 100 00Prague 10, Czech Republic
Full list of author information is available at the end of the article
© 2015 Jiroutková et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Jiroutková et al. Critical Care (2015) 19:448
DOI 10.1186/s13054-015-1160-x

key proteins of the respiratory chain and assessed
mitochondrial function in the cytosolic context by high-
resolution respirometry in fresh skeletal muscle homoge-
nates [8].
Methods
Overview of study design
We performed vastus lateralis muscle biopsies in eight
patients with protracted critical illness and in eight
metabolically healthy control subjects undergoing hip
replacement surgery. From the sample (150–200 mg) we
prepared a homogenate, which was divided into two
parts: the first part was immediately used for respirome-
try analysis (Protocols 1 and 2), whilst the second part
was mixed in 1:1 with a lysis buffer and protease inhibi-
tor, deeply frozen and kept at –80 °C for subsequent
analysis of respiratory complex individual concentrations
(by western blot) and activities (by spectrophotometry).
Study subjects
Study subjects (age 66.6 ± 6.6 years, proportion male/
female 5/3, body mass index (BMI) 27.1 ± 5.4) were
recruited in a general ICU with 22 ventilated beds and
a 10-bed medical ICU at Kralovske Vinohrady Univer-
sity Hospital in Prague. Control subjects (age 61.4 ±
15.8 years, proportion male/female 4/4, BMI 26.6 ± 3.1)
were age-matched metabolically healthy patients under-
going elective hip replacement surgery for degenerative
disease, in the Department of Orthopedics of the same
hospital. All patients gave prospective informed con-
sent. In patients unable to sign the form due to muscle
weakness, the consent procedure was witnessed and
assented by the next of kin. The University Hospital
Ethical Review Board reviewed both the protocol and
the consent form and approved the study. We included
patients who had been ventilator-dependent for more
than 2 weeks and scored <48 points in the Medical
Research Council (MRC) score of muscle weakness [9]
(scale 0–60 points where 0 means most severe weak-
ness and 60 normal muscle power, an objective meas-
ureofmuscleweakness).Weexcludedpatientswith
pre-existing neurological disease, those with severe co-
agulopathy (platelets <50 G/L or international normal-
ized ratio (INR) >1.5) precluding muscle biopsy and
patients receiving steroids in higher than substitution
doses. Out of 22 eligible ICU patients approached, only
8 consented for muscle biopsy.
Characteristics of study subjects are given in Table 1.
Further details about the clinical course of their critical
illness preceding the biopsy, including nutrition and glu-
cose control, are in Additional file 1.
Muscle biopsies and sample treatment
Unless stated otherwise, all chemicals were obtained
from Sigma-Aldrich (St Louis, MO). For a full detailed
description of the methods, a list of media and buffers
and the step-by-step protocol, see Additional file 1.
Muscle biopsies were taken by 5 mm Bergstrom nee-
dle [10] from the vastus lateralis muscle approximately
10 cm above the knee. In order to minimize patients’
discomfort, biopsies from ICU patients were taken under
brief general anesthesia, which was required for a rou-
tine clinical procedure unrelated to the study (e.g., chan-
ging a central line). The sample was collected into 5 mL
of relaxing solution BIOPS containing 10 mM CaK
2
-
EGTA, 7.23 mM K
2
-EGTA, 20 mM imidazole, 20 mM
taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.56 mM
MgCl
2
, 5.77 mM ATP and 15 mM phosphocreatine
adjusted to pH 7.1. The biopsies were kept on ice until
further processing.
High-resolution respirometry on skeletal muscle
homogenates
High-resolution respirometry uses polarographic meas-
urement of oxygen consumption by a Clark electrode.
This method has been adapted to tissue homogenates
Table 1 Study subject characteristics
Subject Diagnosis Age APACHE II Biopsy day MRC score LOS-ICU, days Survived
1 Septic shock, bronchopneumonia 70 22 15 20 34 N
2 Aspiration pneumonia 80 15 29 23 71 Y
3 Sepsis 60 31 40 25 92 N
4 Cardiogenic shock 65 27 41 4 45 Y
5 CHF + CAP 68 10 27 8 30 Y
6 Chest trauma + HAP 62 14 17 18 48 Y
7 CABG, GI bleed 68 23 25 16 43 Y
8 CAP 60 15 30 23 35 Y
Mean ± SD 67 ± 7 20 ± 7 28 ± 9 17 ± 8 50 ± 21 -
Survival means survival to discharge from hospital. APACHE II Acute physiology and chronic health evaluation II score; MRC Medical Research Council score of muscle
power, LOS ICU length of stay in intensive care, CHF congestive heart failure, CAP community-acquired pneumonia, CABG coronary artery bypass grafting,
HAP hospital-acquired pneumonia, GI gastrointestinal, Nno, Yyes
Jiroutková et al. Critical Care (2015) 19:448 Page 2 of 9

[11] including those obtained from human skeletal
muscle needle biopsy samples and validated against
permeabilized muscle fibers [12] and isolated mitochon-
dria [8]. In brief, connective tissue, fat and blood vessels
were gently removed; the skeletal muscle fibers were
dried by gauze and weighed on a calibrated scale (= wet
weight, Ww). After addition of K media (1 mL/100 mg
of muscle Ww), muscle fibers were homogenized by 4–6
strokes in the Elvhjem-Potter teflon/glass homogeniser.
Respirometry was performed at 30 °C without preoxy-
genation with 0.2 mL of 10 % homogenate and 1.9 mL
of K media in the respirometer Oxygraph 2 K (Oroboros
Instruments, Innsbruck, Austria). K medium contains
TrisHCl (10 mM), KCl (80 mM), MgCl
2
(3 mM),
KH
2
PO
4
(5 mM), ethylenediaminetetraacetic acid
(EDTA) (1 mM), BSA (0.5 mg/ml) and water at pH 7.4.
Oxygen concentrations were kept above a predetermined
K90 at all times (See Figure S1 in Additional file 1). Two
assays were performed in parallel in two chambers of
the respirometer by serial addition of substrates and
inhibitors with a Hamilton pipette.
Protocol 1 (see Fig. 1): analysis of global mitochondrial
functional indices on homogenates was performed by
serial addition of malate (2.5 mM) + glutamate (15 mM),
ADP (1 mM), cyt c (10 μM), succinate (10 mM), oligo-
mycin (1 μM), FCCP (0.7 μM), and antimycin A (4 μM).
Non-mitochondrial respiration was oxygen consumption
measured after addition of antimycin A and subtracted
from other values. Capacity of oxidative phosphorylation
(OXPHOS, or 3p respiration) was oxygen consumption
rate when substrates for both complexes I (malate,
glutamate) and II (succinate), abundant ADP and cyto-
chrome c were present. The respiratory chain capacity
(state 3u) was measured after uncoupling with FCCP.
ATP synthesis rate was defined as the decrease in
oxygen consumption after addition of oligomycin when
substrates for complex I and II were present. The
addition of cytochrome c allows for testing preservation
of outer mitochondrial membrane integrity during hom-
ogenisation, with values <20 % considered acceptable
[13]. In our subjects the values were 13 ± 6 % in ICU
and 11 ± 8 % in control patients.
Protocol 2: functional analysis of individual respiratory
complexes. We used serial additions (final concentra-
tions in respirometry chamber) of malate (2.5 mM) and
glutamate (15 mM); ADP (1 mM); cytochrome c
(10 μM) rotenone (3 μM), succinate (10 mM), malonate
(5 mM), glycerol-3-phosphate (5 mM), antimycin A
(4 μM), ascorbic acid (10 mM) and tetramethyl-p-
phenylenediamine (TMPD, 200 μM) and KCN (1 mM).
Complex I activity was calculated as the decrease in oxy-
gen consumption after its inhibitor rotenone, complex II
activity as a decrease after addition of malonate. Com-
plex III/glycerol-3-phosphate dehydrogenase (GPDH)
activity was determined as an increase of oxygen con-
sumption after addition of glycerol-3-phosphate after
both complexes I and II had been inhibited by rotenone
and malonate, respectively. Complex IV activity was
measured as the increase of oxygen consumption after
addition of complex IV substrates ascorbate/TMPD after
complex III had been inhibited by antimycin A. See
Fig. S2, Additional file 1.
Spectrophotometric analysis of individual activities of
respiratory complexes has been described in detail
elsewhere [14]. In brief, frozen sample was thawed and
homogenized and then exposed to three further cycles
of rapid freezing thawing. Complex I assay was per-
formed in an assay mixture composed of 25 mM potas-
sium phosphate, 3.5 g/l BSA, 2 mM EDTA, 60 μM
dichlorophenollindophenol (DCIP), 70 μM decylubiqui-
none, 1 μM antimycin A and 0.2 mM reduced nicotina-
mide adenine dinucleotide (NADH), pH 7.8. Changes in
absorbance were followed at 600 nm. Rotenone sensitive
activity was calculated by subtracting the activity of wells
with 10 μM rotenone. Complex II activity was measured
in an assay mixture containing 80 mM potassium
Fig. 1 An example of high-resolution respirometry assay in a homogenate of skeletal muscle, Protocol 1. Solid line represents oxygen consumption rate,
dashed line oxygen concentration. Mal/Glu malate/glutamate, suc succinate, oligo oligomycin, FCCP uncoupler, AA antimycin A
Jiroutková et al. Critical Care (2015) 19:448 Page 3 of 9

phosphate, 1 g/l BSA, 2 mM EDTA, 10 mM succinate,
80 μM DCIP, 50 μM decylubiquinone, 1 μM antimycin
A and 3 μM rotenone, pH 7.8. Changes in absorbance
were followed at 600 nm. Malonate sensitive activity was
calculated by subtracting the activity of wells with
20 mM malonate. Complex III activity was measured in
an assay mixture containing 50 μM ferricytochrome c,
25 mM potassium phosphate, 4 mM sodium azide,
0.1 mM EDTA, 0.025 % Tween20 and 50 μM decylubi-
quinol, pH 7.4. Changes in absorbance were followed at
550 nm. Antimycin A sensitive activity was calculated by
subtracting the activity of wells with 10 μM antimycin
A. Complex IV activity was measured in an assay buffer
containing 30 mM potassium phosphate and 25 μMof
freshly prepared ferrocytochrome c, pH 7.4. Changes in
absorbance were followed at 550 nm. The absorbance of
samples oxidized with 10 μl of 0.5 M potassium hexa-
cyanoferrate (III) was subtracted from all measurements,
and then the natural logarithm absorbance was plotted
against time and compared to untreated control. Citrate
synthase activity was measured using a commercial kit
from Sigma, as per manufacturer’s instructions [14].
Western blots
Samples containing 6 μg of proteins were mixed with
sample buffer and denatured by heating at 45 °C for
15 minutes. SDS-PAGE and western blotting were per-
formed as described previously [15]. Briefly, proteins
were separated on 12 % polyacrylamide gels at 120 V
and then blotted onto a 0.2 μm nitrocellulose membrane
(Protran BA83, Schleicher-Schuell, Dassel, Germany) for
3 h at 0.25 A. The membranes were blocked in 5 %
weight/volume BSA in Tris-buffered saline for 30 mi-
nutes at room temperature. The washed membranes
were probed with primary antibody cocktail Anti-human
Total OxPhos Complex Kit at 4 °C overnight (dilution
1:175, # 458199, Life Technologies), containing primary
antibodies against complex I (18 kDa), complex II
(29 kDa), complex III (core 2; 48 kDa), complex IV
(cytochrome c oxidase (COX) II subunit, 22 kDa) and
F
1
F
O
ATPase (F1α; 45 kDa) subunits. After washing, the
membranes were incubated for 2 h at room temperature
with mouse horseradish peroxidase-conjugated second-
ary antibody (dilution 1:6600; Santa Cruz Biotechnology,
Santa Cruz, CA, USA). Protein bands were visualized
with an enhanced chemiluminescence detection system
(Thermo Fisher Scientific, Rockford, IL, USA) using
Carestream Gel Logic 4000 PRO Imaging System
(Carestream Health, New Haven, CT, USA). To demon-
strate equal loading, the membrane was stripped and re-
probed with anti-GAPDH antibody (dilution 1:1000, #
ab9485, Abcam, Cambridge, UK). Densitometry was per-
formed using the Carestream v5.2 program (Carestream
Health). Data were normalized to glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) and referenced to an
internal standard (a control patient sample was present on
every immunoblot).
Statistics
Data are presented as median (interquartile range). The
Man–Whitney Utest was used for all comparisons. Sta-
tistica 8.0 (StatSoft Inc., USA) was used for all calcula-
tions and p <0.05 was considered statistically significant.
Results
Relative content of mitochondrial proteins
In ICU patients compared to controls, there was a
significant reduction of core 2 subunit of complex III
(median content in ICU patients was approximately
38 % of that in controls, p= 0.02) and COX2 subunit of
complex IV (approximately 26 %, p<0.01). No differences
were detected in subunits of F
1
F
O
ATPase (approximately
109 %, p= 0.89) or complex II (approximately 90 %,
p=0.99).(seeFig.2).Wewereunabletodetermine
the content of subunits of complex I (the signals
were bellow detection limits in both ICU and control
patients).
Global indices of mitochondrial function in skeletal
muscle homogenates (Protocol 1)
In the skeletal muscle of patients with protracted critical
illness (ICU) compared to control subjects (control),
there was a reduction in citrate synthase (CS) activity
per muscle wet weight (median 0.25 (IQR 0.16–0.28) vs
0.34 (IQR 0.28–0.43) nkat/mg Ww, p= 0.03). In keeping
with this, the capacity of OXPHOS and of the respiratory
chain were significantly reduced in ICU patients (approxi-
mately 54 % and 52 % of that in controls, p<0.01
and p= 0.03) when expressed per muscle wet weight.
OXPHOS normalized to muscle wet weight was
correlated with the activity of CS (r
2
=0.53, p= 0.01),
the content of COX II subunit of respiratory complex IV
(r
2
=0.39, p= 0.03) and there was a trend towards a cor-
relation to core 2 subunit of complex III (r
2
=0.29,
p= 0.06), but no relations at all were seen to con-
centrations of complex II (r
2
=0.04, p=0.50) or F
1α
subunit of F
1
F
O
ATP a se (r
2
=0.02, p= 0.67).
After adjustment to CS activity, the differences in
mitochondrial functional indices between ICU patients
and control subjects disappeared (see Table 2). Of note,
there was no difference in the degree of uncoupling of
inner mitochondrial membrane between ICU patients
and controls.
Analysis of function of individual respiratory complexes
Protocol 2: by using sequential addition of substrates
and specific inhibitors of the respiratory chain com-
plexes, we were able to determine maximum electron
Jiroutková et al. Critical Care (2015) 19:448 Page 4 of 9

fluxes through them. Oxygen consumption rates were
adjusted to the activity of CS (see Fig. 3, upper row).
Functional capacity of complexes I and IV were not
different between ICU patients and the control group.
Surprisingly, the capacity of complex II was demonstrated
in the ICU group to be approximately 300 % of the cap-
acity in controls (p<0.01). The capacity of complex III/
GPDH was also significantly (p<0.01) higher in ICU pa-
tients as compared to controls. Very similar results were
obtained when the individual activity of respiratory com-
plexes was measured by spectrophotometry (Fig. 3).
Last we asked whether there is a relationship between
the capacity of OXPHOS (or state 3p as determined in
Protocol 1) and specific functional capacity of individual
respiratory complexes (as determined in Protocol 2).
Complex I (r
2
= 0.33, p= 0.04), and even more strongly
complex IV (r
2
= 0.65, p<0.01) correlated to OXPHOS,
whilst complexes II and III/GPDH did not (r
2
= 0.08,
p=0.36 and r
2
= 0.12, p= 0.28, respectively). See Fig.
S3 in the Supplementary material.
Discussion
This study is the first to demonstrate mitochondrial dys-
function in skeletal muscle of patients with protracted
critical illness. In the skeletal muscles of these patients
we observed approximately 50 % reduction in the ability
to synthetize ATP by aerobic phosphorylation per mg of
muscle wet weight (OXPHOS/W
w
) which correlated
Fig. 2 Concentrations of functional subunits of respiratory complexes in arbitrary units and an example of an immunoblot membrane. Data are
presented as medians, vertical bars represent interquartile ranges. GAPDH glyceraldehyde 3-phosphate dehydrogenase, COX cytochrome coxidase
Jiroutková et al. Critical Care (2015) 19:448 Page 5 of 9

Table 2 Mitochondrial functional indices measured by high-resolution respirometry in homogenates
Parameter Per muscle wet weight (pmol/s.mg Ww) Per CS activity (pmol.nkat-1.s-1)
ICU (n = 7) Control (n = 8) PICU (n = 7) Control (n = 8) P
OXPHOS (3p) 7.6 (5.0–8.8)
*
13.9 (11.3–17.9) <0.01 31 (28–36)
*
37 (32–74) 0.15
RC capacity (3u) 8.6 (6.7–10.5) 16.4 (13.0–20.6) 0.03 41 (37–44) 42 (37–98) 0.46
Non-mito OCR 0.8 (0.6–1.5) 0.8 (0.6–1.3) 0.91 4 (3–5) 2 (1–4) 0.16
F
1
F
o
ATPase Absolute 6.1 (4.8–7.6)
*
12.6 (9.2–13.0) <0.01 26 (26–30)
*
33 (29–49) 0.46
% OXPHOS 81 (77–83)
*
84 (80–89) 0.36 81 (77–83)
*
84 (80–89) 0.36
Proton leak Absolute 1.3 (1.0–1.4)
*
2.2 (1.3–3.6) 0.10 8 (5–9)
*
7(4–11) 0.95
% OXPHOS 19 (17–23)
*
16 (11–20) 0.36 19 (17–23)
*
16 (11–20) 0.36
Complex I 4.8 (4.0–6.1) 6.7 (5.5–8.6) 0.19 23 (22–35) 23 (18–26) 0.35
Complex II 4.6 (2.9–6,5) 1.5 (0.8–3.8) 0.06 23 (20–28) 8 (3–14) <0.01
Complex III/ GPDH 1.5 (1.1–1.9) 0.8 (0.4–1.3) 0.12 7.4 (6.0–9.3) 1.8 (1.2–3.9) <0.01
Complex IV 15.5 (13.0–19.5) 19.7 (15.3–27.5) 0.30 88 (69–99) 49 (40–113) 0.12
Data presented as median (interquartile range), pvalue as per Mann–Whitney Utest. *N = 5 for ICU patients. GPDH glycerol-3-phosphate dehydrogenase, Non-mito
OCR non-mitochondrial oxygen consumption rate, OXPHOS oxidative phosphorylation, RC respiratory chain
Fig. 3 Activity of individual respiratory complexes adjusted to mitochondrial content (citrate synthase activity) measured by two independent methods.
Upper row complex activity in cytosolic context determined by high-resolution respirometry in skeletal muscle homogenates. Lower row
spectrophotometric analysis of the activity of individual respiratory complexes. Lines represent medians
Jiroutková et al. Critical Care (2015) 19:448 Page 6 of 9

with the concentration of depleted complex IV. Complex
III was also depleted, unlike complexes II and V. When
OXPHOS was adjusted to citrate synthase activity
(OXPHOS/CS), the differences between ICU patients
and control subjects disappeared and OXPHOS/W
w
strongly correlated with citrate synthase activity. The ob-
vious interpretation of these results is that mitochondria
are depleted in ICU patients, whilst complexes II and V
are relatively abundant in remaining functional mito-
chondria. A similar disproportionality of the concentra-
tions of respiratory complexes has been described in
skeletal muscle during aging [16] and oxidative stress
[17]. Even though citrate synthase activity is widely used
as a marker of mitochondrial content [2, 18–20], it may
become a subject of oxidative damage [21] and therefore
it may not reliably reflect the mitochondrial density.
Because we have not used an alternative method of
measuring mitochondrial content (e.g., electron micros-
copy), we cannot say whether the depletion of com-
plexes III and IV occurred in isolation or as part of
mitochondrial depletion. It is the concentration of the
depleted complex IV (and possibly complex I) that was
limiting for the mitochondrial function, in keeping with
data of Levy [22], who demonstrated the relation of
complex IV dysfunction to bioenergetics failure in acute
sepsis. Contrary to our hypothesis, there was no sign of
increased mitochondrial uncoupling in ICU patients.
In order to explore the functional capacity of individ-
ual complexes, we performed a respirometry protocol in
which we used specific substrates and inhibitors of indi-
vidual complexes. If expressed per muscle wet weight
(Table 2), we saw a trend towards increase in functional
capacity of respiratory complexes II and III, whilst that
of complexes I and IV tended to be non-significantly
reduced to approximately 70 % of values seen in control
subjects, and correlated with OXPHOS. After adjust-
ment for citrate synthase activity, complexes II and III
were increased significantly (threefold and twofold
respectively, p<0.01) and complexes I and IV were not
different (Fig. 3). High-resolution respirometry measures
the changes in oxygen consumption in fresh intact tissue
homogenates after addition of respiratory substrates and
inhibitors [11]. The sample contains intact mitochondria
in a cytosolic context and it is believed that this ap-
proach better reflects physiological alterations occurring
in vivo [23]. The technique has been calibrated against
permeabilized muscle fibers [12] and isolated mitochon-
dria [8]. When using this method for measuring the
functional capacity of individual complexes one must
bear in mind that the rate-limiting step can in theory ap-
pear downstream of the complex that is being analyzed.
Complexes III and IV are under physiological conditions
able to accommodate the flux of electrons from both
complexes I and II and it is therefore unlikely that they
become rate-limiting when fed by electrons from either
complex I or II in isolation. For testing complex III we
used glycerol-3-phosphate as a substrate whilst com-
plexes I and II had been blocked. By doing so we
avoided the risk of downstream limitation (i.e., at com-
plex IV), but on the other hand, the rate-limiting step
may be at the level of GPDH, which is functionally a
part of the glycerol phosphate shuttle rather than the
respiratory chain.
With these limitations of respirometry in mind, we re-
peated the measurements of individual complex activities
by a different technique. Classical spectrophotometry is a
well-established method [2, 3, 18], which assesses the
activities of respiratory complexes by using artificial
complex-specific substrates after the organelle structure
has been destroyed by repeated freezing and thawing. This
means that the measured activity of each complex is
independent of the functionality of other complexes. As
demonstrated in Fig. 3, both methods gave very similar
results and confirmed the increased functional capacity
of complexes II and III/GPDH in the critically ill as
compared to control subjects.
Complex II (succinate dehydrogenase) normally drives
electrons from succinate oxidation to fumarate in the
citric acid cycle (CAC) via flavin adenine dinucleotide
(FAD) to the respiratory chain. CAC itself is heavily
dependent on reoxidation of NADH by complex I as it
produces three molecules of NADH per one molecule of
FADH
2
. Eventual increase in NADH/NAD+ ratio inhibits
CAC. Similarly, aerobic glycolysis produces 2NADH/mol-
ecule of glucose during the conversion to pyruvate and a
further 2NADH by converting pyruvate to acetyl-CoA,
which is oxidised in CAC. However, during oxidation of
fatty acid and carbon skeletons of branched chain amino
acids, reduced coenzymes FADH
2
and NADH are pro-
duced in a 1:1 ratio. Of all catabolic pathways, fatty acid
oxidation is thus least dependent on the functionality of
complex I. In the acute phase of critical illness complex I
seems to be predominantly impaired [2] and upregulation
of complex II at a later stage can be a compensatory
response or an attempt to bypass dysfunctional complex I.
Insulin resistance is a well-known feature of critical illness
[24, 25] and it has been shown that GLUT-4 dependent
transport is dysfunctional in patients with ICUAW (weak-
ness developing in a critically ill patient without an identi-
fiable cause other than nonspecific inflammation) [26] and
pyruvate dehydrogenase is inhibited [27]. Skeletal muscle
in protracted critical illness thus may suffer from starva-
tion of carbohydrate-derived substrate for CAC. On the
contrary, free fatty acids are elevated in the critically ill
[24, 28] and intracellular lipid droplets accumulate early
in diaphragmatic and biceps muscle in brain-dead donors
[18]. Branched-chain amino acids (BCAA) derived from
muscle protein degradation are deaminated in skeletal
Jiroutková et al. Critical Care (2015) 19:448 Page 7 of 9

muscle and their carbons are oxidized in a similar way to
fatty acid oxidation. Relative upregulation of complex II in
the context of mitochondrial dysfunction may thus repre-
sent an adaptive response to insulin resistance [29] and
preferential oxidation of lipids and BCAA over carbohy-
drates. Glycerol-3-phosphate can be formed from glycerol
derived from lipolysis [30], and it requires respiratory
complexes distal to complex I to be converted to
glyceraldehyde-3-phosphate [31], a glycolytic intermediate.
Upregulation of complex III/GPDH seen in our ICU pa-
tients may reflect the increase in intracellular lipid turnover
in the skeletal muscle of these patients.
However, the lack of correlation between OXPHOS
and both functional capacities and relative abundance of
complexes II and III/GPDH suggests that they may play
other functions, which are not directly related to aerobic
ATP production. It has been recently shown that cells
accumulate succinate during hypoxia [32–34] or inflam-
mation [35]. When oxygenation is restored, rapid re-
oxidation of succinate produces electron flux, which
downstream complexes are unable to absorb, and which
is redirected backwards to complex I, generating exces-
sive amounts of reactive oxygen species [36, 37]. Relative
redundancy of the activity of complexes II and III over
complex I observed by us in protracted illness could be
an adaptation against cell damage when intracellular
succinate levels are fluctuating.
Indeed our study has many limitations. First, our data
are derived from a small group of highly selected
subjects. We found it very difficult to consent patients
for the biopsy in this non-therapeutic study. With such
a small number of subjects there is always a risk of type
II error, i.e., that we were unable to detect changes that
were present. High inter-individual variability in the
concentration and functionality of respiratory com-
plexes (see Fig. 3) is well-known [2, 22], and further
complicates the interpretation of data. Biopsies were
performed in ICU patients who had been ventilator-
dependent for more than 2 weeks (mean 28 days) and
suffered from muscle weakness. We have selected this
cohort of patients with muscle dysfunction in order to
maximize the chances of observing any alteration of
bioenergetics in a non-respiratory muscle, which seems
to be less affected, even in the acute phase of critical
illness, when compared to the diaphragm [18, 38] or
intercostal muscles [3]. As a result, it remains unclear
whether the changes in mitochondrial metabolism
described above are consequences of prolonged immo-
bility [39–41], the critical illness, or whether they occur
only in patients who are weak. Of note, our control
subjects were ambulatory elective hip surgery patients
and it is unknown whether their potentially reduced
mobility affected the mitochondrial function of skeletal
muscle. In light of this, our pilot study should be
treated as a proof-of-concept study and the results
interpreted with caution.
Conclusions
In conclusion, we have demonstrated mitochondrial
dysfunction in the quadriceps muscle of patients with
protracted critical illness compared to metabolically
healthy age-matched control patients undergoing hip
replacement surgery. There was approximately 50 %
reduction in the capacity for aerobic ATP synthesis
per mg of muscle wet weight, in correlation with sig-
nificant reductions in functional subunits of com-
plexes III and IV. When accounting for the activity of
citrate synthase, which we used as a marker of mito-
chondrial content, there was no difference in global
mitochondrial functional indices. We have shown a
significant increase in the functional capacity of com-
plexes II and III/GPDH. This can be possibly ex-
plained by metabolic adaptation to insulin resistance
or succinate fluctuation, but exploring these hypoth-
eses warrants further studies.
Additional file
Additional file 1: Supplementary material. (DOCX 115 kb)
Abbreviations
ATP: adenosine triphosphate; BCAA: branched-chain amino acids; BMI: body
mass index; BSA: bovine serum albumin; CABG: coronary artery bypass
grafting; CAC: citric acid cycle; CAP: community-acquired pneumonia;
CHF: congestive heart failure; COX: cytochrome coxidase; CS: citrate
synthase; Cyt c: cytochrome c; DCIP: dichlorophenollindophenol;
EDTA: ethylenediaminetetraacetic acid; FAD: flavin adenine dinucleotide;
FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone;
GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GPDH: glycerol-3-
phosphate dehydrogenase; HAP: hospital-acquired pneumonia;
ICUAW: Intensive Care Unit - acquired weakness; kDa: kiloDalton; LOS
ICU: length of stay in intensive care; MRC: Medical Research Council score of
muscle power, NAD
+
, nicotinamide adenine dinucleotide; OXPHOS: oxidative
phosphorylation; TMPD: N,N,N',N'-tetramethyl-p-phenylenediamine;
Ww: wet weight.
Competing interests
The authors declare no competing interests.
Authors’contributions
KJ processed muscle samples, participated in the respirometry analysis and
contributed to the study design. AK and JZ helped to design the study,
processed muscle samples, performed (together with MK) the respirometry
analyses. ME and JT (together with AK) performed spectrophotometric analysis
of respiratory complexes. VD, MF and JG obtained informed consents and
performed the biopsies, whilst VFN and JK carried out the western blots. FD
conceived of the study, participated in its coordination and performed the
statistical analysis. All authors wrote their parts of the manuscript, revised the
first draft and then read and approved the final version of the manuscript.
Acknowledgements
The study was supported from grants IGA NT 12319 and PRVOUK P31
(coordinator: Prof. Michal Andel). We thank all volunteers who participated in
the study and Dr Paul James for proofreading.
Jiroutková et al. Critical Care (2015) 19:448 Page 8 of 9

Author details
1
Laboratory of Bioenergetics, Third Faculty of Medicine, Charles University in
Prague, Ruská 87, Prague 100 00Prague 10, Czech Republic.
2
Department of
Internal Medicine II, Kralovske Vinohrady University Hospital, Prague, Czech
Republic.
3
Department of Orthopaedic Surgery, Kralovske Vinohrady
University Hospital, Prague, Czech Republic.
4
Department of Anaesthesia and
Intensive Care, Kralovske Vinohrady University Hospital, Prague, Czech
Republic.
5
Department of Cell and Molecular Biology & Center for Research
of Diabetes, Metabolism and Nutrition, Third Faculty of Medicine, Charles
University in Prague, Prague, Czech Republic.
6
Adult Intensive Care Unit,
Queen’s Medical Centre, Nottingham University Hospital NHS Trust,
Nottingham, UK.
Received: 8 September 2015 Accepted: 6 December 2015
References
1. Takasu O, Gaut JP, Watanabe E, To K, Fagley RE, Sato B, et al. Mechanisms of
cardiac and renal dysfunction in patients dying of sepsis. Am J Respir Crit
Care Med. 2013;187:509–17.
2. Brealey D, Brand M, Hargreaves I, Heales S, Land J, Smolenski R, et al.
Association between mitochondrial dysfunction and severity and outcome
of septic shock. Lancet. 2002;360:219–23.
3. Fredriksson K, Hammarqvist F, Strigård K, Hultenby K, Ljungqvist O,
Wernerman J, Rooyackers O. Derangements in mitochondrial metabolism in
intercostal and leg muscle of critically ill patients with sepsis-induced
multiple organ failure. Am J Physiol Endocrinol Metab. 2006;291:E1044–50.
4. Carré JE, Orban J-C, Re L, Felsmann K, Iffert W, Bauer M, et al. Survival in
critical illness is associated with early activation of mitochondrial biogenesis.
Am J Respir Crit Care Med. 2010;182:745–51.
5. Garrabou G, Morén C, López S, Tobías E, Cardellach F, Miró O, Casademont
J. The effects of sepsis on mitochondria. J Infect Dis. 2012;205:392–400.
6. Vanhorebeek I, De Vos R, Mesotten D, Wouters PJ, De Wolf-Peeters C, Van
den Berghe G. Protection of hepatocyte mitochondrial ultrastructure and
function by strict blood glucose control with insulin in critically ill patients.
Lancet. 2005;365:53–9.
7. Japiassú AM, Santiago AP, d’Avila JC, Garcia-Souza LF, Galina A, Castro Faria-
Neto HC, et al. Bioenergetic failure of human peripheral blood monocytes
in patients with septic shock is mediated by reduced F1Fo adenosine-5'-
triphosphate synthase activity. Crit Care Med. 2011;39:1056–63.
8. Ziak J, Krajcova A, Jiroutkova K, Nemcova V, Dzupa V, Duska F. et al.
Assessing the function of mitochondria in cytosolic context in human
skeletal muscle: Adopting high-resolution respirometry to homogenate of
needle biopsy tissue samples. Mitochondrion. 2015;21:106–12.
9. Edwards RHT. Muscle fatigue. Postgrad Med J. 1975;51:137–143. http://pmj.
bmj.com/content/51/593/137.
10. Hayot M, Michaud A, Koechlin C, Caron MA, Leblanc P, Préfaut C, Maltais F.
Skeletal muscle microbiopsy: a validation study of a minimally invasive
technique. Eur Respir J. 2005;25:431–40.
11. Pecinová A, Drahota Z, Nůsková H, Pecina P, Houštěk J. Evaluation of basic
mitochondrial functions using rat tissue homogenates. Mitochondrion.
2011;11:722–8.
12. Larsen S, Kraunsøe R, Gram M, Gnaiger E, Helge JW, Dela F. The best
approach: Homogenization or manual permeabilization of human skeletal
muscle fibers for respirometry? Anal Biochem. 2014;446:64–8.
13. Schuh RA, Jackson KC, Khairallah RJ, Ward CW, Spangenburg EE. Measuring
mitochondrial respiration in intact single muscle fibers. AJP. 2012;302:R712–
9.
14. Trnka J, Elkalaf M, Andel M. Lipophilic triphenylphosphonium cations inhibit
mitochondrial electron transport chain and induce mitochondrial proton
leak. PLoS ONE. 2015;10:e0121837–14.
15. Němcová-Fürstová V, James RF, KovářJ. Inhibitory effect of unsaturated fatty
acids on saturated fatty acid-induced apoptosis in human pancreatic cells:
activation of caspases and ER stress induction. Cell Physiol Biochem. 2011;
27(5):525–38.
16. Hepple RT, Baker DJ, Kaczor JJ, Krause DJ. Long-term caloric restriction
abrogates the age-related decline in skeletal muscle aerobic function. FASEB
J. 2005;19(10):1320–2.
17. Crane JD, Abadi A, Hettinga BP, Ogborn DI, MacNeil LG, Steinberg GR, et al.
Elevated mitochondrial oxidative stress impairs metabolic adaptations to
exercise in skeletal muscle. PLoS One. 2013;8(12):e81879.
18. M, Jung B, Liang F, Azuelos I, Hussain S, Goldberg P, et al. Mitochondrial
dysfunction and lipid accumulation in the human diaphragm during
mechanical ventilation. Am J Respir Crit Care Med. 2012;186:1140–9.
19. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, et al.
Biomarkers of mitochondrial content in skeletal muscle of healthy young
human subjects. J Physiol. 2012;590(Pt 14):3349–60.
20. Wibom R, Hultman E, Johansson M, Matherei K, Constantin-Teodosiu D,
Schantz PG. Adaptation of mitochondrial ATP production in human skeletal
muscle to endurance training and detraining. J Appl Physiol. 1992;73:2004–10.
21. Chepelev NL, Bennitz JD, Wright JS, Smith JC, Willmore WG. Oxidative
modification of citrate synthase by peroxyl radicals and protection with
novel antioxidants. J Enzyme Inhib Med Chem. 2009;24(6):1319–31.
22. Levy RJ, Deutschman CS. Cytochrome c oxidase dysfunction in sepsis. Crit
Care Med. 2007;35:S468–75.
23. Barrientos A, Fontanesi F, Diiaz F. Evaluation of the Mitochondrial
Respiratory Chain and Oxidative Phosphorylation System Using
Polarography and Spectrophotometric Enzyme Assays. Hoboken, NJ, USA:
John Wiley & Sons, Inc; 2001.
24. Cree MG, Wolfe RR. Postburn trauma insulin resistance and fat metabolism.
Am J Physiol Endocrinol Metab. 2008;294:E1–9.
25. Bakalar B, Duska F, Pachl J, Fric M, Otahal M, Pazout J, Andel M. Parenterally
administered dipeptide alanyl-glutamine prevents worsening of insulin
sensitivity in multiple-trauma patients. Crit Care Med. 2006;34(2):381–7.
26. Weber-Carstens S, Schneider J, Wollersheim T, Assmann A, Bierbrauer J,
Marg A, et al. Critical illness myopathy and GLUT4. Am J Respir Crit Care
Med. 2013;187:387–96.
27. Vary TC. Sepsis-induced alterations in pyruvate dehydrogenase complex
activity in rat skeletal muscle: effects on plasma lactate. Shock. 1996;6:89–94.
28. Duska F, Fric M, Waldauf P, Pazout J, Andel M, Mokrejs P, Tůma P, Pachl J.
Frequent intravenous pulses of growth hormone together with glutamine
supplementation in prolonged critical illness after multiple trauma: Effects
on nitrogen balance, insulin resistance, and substrate oxidation. Crit Care
Med. 2008;36(6):1707–13.
29. Lee JS. Saturated, but not n-6 polyunsaturated, fatty acids induce insulin
resistance: role of intramuscular accumulation of lipid metabolites. J Appl
Physiol. 2006;100:1467–74.
30. Watford M. Functional glycerol kinase activity and the possibility of a major role
for glyceroneogenesis in mammalian skeletal muscle. Nutr Rev. 2000;58:145–8.
31. Mráček T, Drahota Z, Houštěk J. The function and the role of the
mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues.
BBA - Bioenergetics. 2013;1827:401–10.
32. Lukyanova LD. Mitochondrial Signaling in Hypoxia. OJEMD. 2013;03:20–32.
33. Chouchani ET, Pell VR, Gaude E, AksentijevićD, Sundier SY, Robb EL, et al.
Ischaemic accumulation of succinate controls reperfusion injury through
mitochondrial ROS. Nature. 2014;515:431–5.
34. O'Neill L. Succinate strikes. Nature. 2014;515:1–2.
35. Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF,
Goel G, et al. Succinate is an inflammatory signal that induces IL-1βthrough
HIF-1α. Nature. 2014;496:238–42.
36. Quinlan CL, Perevoshchikova IV, Hey-Mogensen M, Orr AL, Brand MD. Sites
of reactive oxygen species generation by mitochondria oxidizing different
substrates. Redox Biol. 2013;1:304–12.
37. Chouchani ET, Methner C, Nadtochiy SM, Logan A, Pell VR, Ding S, et al.
Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial
complex I. Nat Med. 2013;19:753–9.
38. Peruchi BB, Petronilho F, Rojas HA, Constantino L, Mina F, Vuolo F, et al.
Skeletal muscle electron transport chain dysfunction after sepsis in rats. J
Surg Res. 2011;167:e333–8.
39. Holloszy JO, Coyle EF. Adaptations of skeletal muscle to endurance exercise
and their metabolic consequences. J Appl Physiol Respir Environ Exerc
Physiol. 1984;56(4):831–8.
40. Berg HE, Dudley GA, Hather B, Tesch PA. Work capacity and metabolic and
morphologic characteristics of the human quadriceps muscle in response to
unloading. Clin Physiol. 1993;13(4):337–47.
41. Hikida RS, Gollnick PD, Dudley GA, Convertino VA, Buchanan P. Structural
and metabolic characteristics of human skeletal muscle following 30 days of
simulated microgravity. Aviat Space Environ Med. 1989;60(7):664–70.
Jiroutková et al. Critical Care (2015) 19:448 Page 9 of 9