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CHAPTER 5 IMPAIRED MITOCHONDRIALDYNAMICS AND DECREASED COMPLEXES INDIAPHRAGM FIBERS OF CRITICALLY ILL PATIENTS

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5. IMPAIRED MITOCHONDRIAL DYNAMICS AND DECREASED COMPLEXESIN DIAPHRAGM FIBERS OF CRITICALLY ILL PATIENTSPleuni E. Hooijman, MSc1; Albertus Beishuizen, MD, PhD2,11; Monique C. de Waard, PhD2;Alaattin Ozdemir11; Armand R. J. Girbes, MD, PhD2; Angelique Spoelstra - de Man, MD,PhD2; Ruud Zaremba1; Michael W. Lawlor, MD, PhD8; Ger J.M. Stienen, PhD1,6; Koen J.Hartemink, MD, PhD5,10; Marinus A. Paul, MD, PhD5; Roberto Bottinelli13,15,16; Maria A.Pellegrino13,14,16; Rob C.I. Wüst, PhD1 and Coen A.C. Ottenheijm, PhD1,17

Departments of 1Physiology, 2Intensive Care, 3Pathology, 4Anesthesiology,5Cardiothoracic Surgery, ICaR-VU, VU University Medical Center, Amsterdam, theNetherlands; 6Faculty of Science, Department of Physics and Astronomy, VUUniversity, Amsterdam, the Netherlands; 7Department of Anesthesiology andOperative Intensive Care, Medical Faculty Mannheim, University of Heidelberg,Germany; 8Division of Pediatric Pathology, Department of Pathology andLaboratory Medicine, Medical College of Wisconsin, Milwaukee, USA;9Department of Integrative Pathophysiology, Universitatsmedizin Mannheim,University of Heidelberg, Germany, 10Department of Surgery, Netherlands CancerInstitute – Antoni van Leeuwenhoek Hospital, Amsterdam, the Netherlands;11Intensive Care, Medisch Spectrum Twente, Enschede, the Netherlands;12Department of Intensive Care Medicine, Pulmonary Diseases, RadboudUniversity Medical Centre, Nijmegen, the Netherlands; 13Department of MolecularMedicine, University of Pavia, Pavia, Italy 14Interuniversity Institute of Myology,University of Pavia, Pavia, Italy; 15Department Fondazione Salvatore Maugeri(IRCCS), Scientific Institute of Pavia, Pavia, Italy ;16Interdepartimental Centre forBiology and Sport Medicine, University of Pavia, 27100, Pavia, Italy; 17Universityof Arizona, Department of Physiology, Tucson, AZ, USA.

Unpublished

IMPAIRED MITOCHONDRIAL DYNAMICS AND DECREASED COMPLEXES IN DIAPHRAGM FIBERS OFCRITICALLY ILL PATIENTS

Abstract

Weakness of the main inspiratory muscle, the diaphragm, may delay weaning frommechanical ventilation, and increase the duration of hospitalization and morbidity ofpatients at the Intensive Care Unit. It remains unknown whether mitochondrialmorphology and function are affected in the diaphragm of critically ill patients. Themitochondrial morphology and function was determined in diaphragm biopsies ofmechanically ventilated critically ill patients (n=28) and compared to control patients(n=27). Critically ill patients had a lower content of mitochondrial complexes, a lowerPGC1-α expression and increased levels of phosphorylated AMPK. Electron microscopysuggested disturbed mitochondrial networks in diaphragm fibers of critically patients,which was supported by protein analyses revealing altered fission/fusion dynamics.Maximal mitochondrial respiratory capacity in permeabilized fibers did, however, notdiffer between groups. Together, these data suggest disturbed metabolic regulation ofmitochondria. We conclude that alterations in metabolic regulation and mitochondrialnetwork dynamics may contribute to diaphragm weakness in critically ill patients.

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Introduction

Mechanically-ventilated critically-ill patients develop weakness of the inspiratorymuscles, as indicated by a reduced pressure generating capacity of the diaphragmmuscle 61–63. Diaphragm weakness contributes to a delay in weaning from mechanicalventilation, and to an increased duration of hospital stay and morbidity in patientsadmitted to the Intensive Care Unit 1,5–7.Recent work from our group suggests that diaphragm weakening in critically ill patientscan be largely accounted for by contractile weakness and atrophy of individualdiaphragm fibers 72,130. The observation that the ubiquitin-proteasome pathway wasactivated in diaphragm fibers suggest a major role for proteolytic pathways in theobserved atrophy 130. To date, the mechanism that trigger proteolysis in diaphragmfibers of mechanically ventilated critically ill patients remains largely unknown.Proteolysis is at least partly regulated by atrophy genes, such as the class O forkheadbox (FoxO) family of transcription factors. AMP-activated protein kinase (AMPK) is anupstream regulator of FoxO and a key metabolic stress sensor. Its activation is knownto cause loss of muscle mass 32–34 and suppression of FoxO reduces the transcription ofkey atrogenes via activation of peroxisome proliferator activated receptor co-activators1 alpha (PGC-1α) 131. PGC-1α is known to induce mitochondrial biogenesis and isinvolved in mitochondrial dynamics (fission/fusion) 132, but the contribution ofmitochondrial function in muscle atrophy is poorly understood.Recent findings suggest that increased mitochondrial fragmentation, i.e. fission, is oneof the triggers that activates the AMPK-FoxO3 signaling axis 27,31,32. Such mitochondrialfission can occur rapidly in response to changes in energy requirements and tometabolic stress 133–137. On the other hand, mitochondrial fusion allows for mixing ofmitochondrial DNA and matrix proteins for optimal quality control and ATP production138. If the individual mitochondria fail to produce ATP during reloading of thediaphragm, for example when respiratory support is decreased or discontinued, thismay impair weaning from mechanical ventilation of critically ill patients.Findings from animal models and brain dead organ donors show that mechanicalventilation induces impaired mitochondrial function in the diaphragm muscle35,73,111,139,140 and that therapies aimed at protecting mitochondrial function ameliorateddiaphragm dysfunction 35,73,141. Whether these impairments in mitochondrial functiontranslate to critically ill patients, who exhibit distinct clinical features, is currentlyunknown.Therefore, in the present study we hypothesized that mitochondria in diaphragmmuscle fibers of mechanically ventilated critically ill patients are subject to (1) alteredfission and fusion dynamics and (2) decreased enzymatic activity. To test thesehypotheses, we obtained diaphragm muscle biopsies from 28 mechanically ventilated

IMPAIRED MITOCHONDRIAL DYNAMICS AND DECREASED COMPLEXES IN DIAPHRAGM FIBERS OFCRITICALLY ILL PATIENTScritically ill patients, and compared these with biopsies obtained from 27 patientsduring thoracic surgery (controls). Mitochondrial morphology was studied by electronmicroscopy. Mitochondrial dynamics and signaling pathways were determined byWestern blotting. Mitochondrial enzyme activity was assessed by respirometry.Results

PatientsControl and critically ill patients were mechanically ventilated for, on average, 1.4±0.1hours and 153±32 hours (p<0.0001), respectively. Control and critically ill patients didnot significantly differ with respect to age (respectively, 59±2years vs. 61±3years, p=0.69), body mass index (respectively, 26.1±0.7 kg.m-2 vs. 24.7±0.7 kg.m-2, p= 0.17) orsex (male/female: 18/9 vs. 13/15, p=0.18).The demographic and clinical characteristics of critically ill patients are shown in Table1a , and those of control patients are shown in Table 1b. Table 1c gives the specificationsof mechanical ventilation of the critically ill patients and in supplementary Table 1 anoverview is provided of the number of patients measured per parameter.Altered mitochondrial structureElectron microscopy micrographs were obtained from 8 controls and 7 patients. Table2 summarizes the findings of the electron microscopy studies and Figure 1 showstypical images of both groups. Abnormal general impression of mitochondria wasobserved in 6/7 critically ill and 3/8 control patients; 4/7 critically ill vs. 2/8 controlshad higher than normal number of mitochondrial aggregates; 3/7 critically ill patientsvs. 1/8 controls had higher number of mitochondria; 3/7 critically ill patients vs. noneof the controls had misalignment of mitochondria. Thus, the electron microscopystudies suggest that in critically ill patients mitochondria loose connection and altertheir location.Legend Table 1. CVA=cerebrovascular accident. PTCA=percutaneous transluminal coronaryangioplasty, SCIAP=sensoric chronic idiopathic axonal polyneuropathy, AVNRT=atrioventricularnodal reentrant tachycardia, DMII=diabetes mellitus type II, PCI=percutaneous coronaryintervention, LAD=left anterior descending artery.

Tabl

e 1a

Cri

tica

lly il

l pat

ient

s–

dem

ogra

phy,

med

ical

his

tory

, rea

son

adm

issi

on IC

U an

d re

ason

surg

ery

whe

re b

iops

y w

as o

btai

ned.

#AgeG

Medical Histor

yReason

admission IC

USurger

y where biops

y was obtained

SepDiedBMI

APII1

48FCOPD

Respiratory f

ailure after VA

TS lobectomy

Re-thoracotom

y: lobectomy

necrotic midd

le lobeNN

18222

67MLV hyp

ertrophy with

decreased EF

Hemorrhagic

shock due to re

troperitoneal

hematomaFin

al closure of ab

dominal woun

d after re-re-

laparotomy

NY2838

367F

Rheumathoid

arthritis, CVA

Septic shock d

ue to intestina

l perforation,

SB resectionR

e-laparotomy:

second look, d

rainage abdom

enYY

22184

53MNone

Thoracic endo

vascular aortic

repair for typ

e B dissection

,hemor

rhagic shock

Thoracotomy

with surgical r

e-evacuation h

ematothorax

NN3029

547F

NoneSevere

trauma

Re-laparotomy

: removal of g

auzeNN

22286

67FHypert

ension, cigare

tte smoker,

thyroid dysfun

ctionGastro

-enteric ische

mia, thrombo

sis of celiac tr

unk and

AMS/ endovas

cular treatmen

tRe-lapa

rotomy for dra

inage abdomin

al abscess

YY2328

770M

NoneEsopha

geal rupture,

Boerhaave syn

dromeThorac

otomyNN

22278

51MNone

Severe traum

a and traumat

ic shock

Re-re-re lapar

otomy for rem

oval gauze

NN2930

946F

Asthma, cigare

tte smoker

Cardiac arrest

due to hemor

rhagic shock

Re-re-laparot

omy for remo

val gauze

NY1937

1052M

NoneSevere

trauma

Relaparotomy

for closing ab

domenYN

292011

81FHypert

ensionSevere

trauma

Thorax surger

y reconstructi

on 2 ribs and

inspection

NY2432

1277F

DMIICecal p

erforation wit

h sepsis. Acut

e kidney failur

eRe-re-la

parotomy for

removal Bogo

ta bagYY

282113

71MCOPD,

colon carcinom

aDuode

nal perforatio

n with sepsis

Re-laparotomy

for cholecyste

ctomy perfora

tion gallbladd

erYY

201914

77FSpinal

cord injury lev

el L4-L5

Gastro-intesti

nal perforatio

nRe-re-l

aparotomy fo

r removal Bog

ota bag en dra

inageYY

232215

74MHypert

ension, atrial

fibrillation

Respiratory f

ailure due to p

neumonia, ICU

staycompli

cated by bowe

l perforation

Re-laparotomy

for evacuation

hematoma

YY2519

1683M

Hypertension

, polyposis, SA

H,rectum

carcinoma

Abdominal sep

sis, Hartmann

procedure

Re-laparotomy

for removalBo

gota bag, hem

icolectomy

YY2435

1768M

COPD, Radiot

herapy for pro

statecarcino

maAortic

dissection, he

matothorax af

ter Bentall pro

cedure,

rib fracture af

ter resuscitat

ionThorac

otomy for fixa

tion of rib frac

turesNN

241618

68FHypert

ension, Nonin

sulin depende

ntdiabete

s. Cerebral in

farction

Respiratory f

ailure due to u

pper airway o

bstruction

complicated b

y type II myoc

ardial infarcti

on.Corona

ry artery bypa

ss graft surger

y and hemistr

umectomy

NY2718

1969F

Hypertension

, smoker, aorti

c valvereplace

mentProgre

ssive heart fai

lure after surg

ery aortic valv

ereplace

ment, perforat

ion of transve

rse colon

Re-laparotomy

for drainage a

scitesNY

341620

22Fnone

Severe traum

aRe-re-r

elaparotomy fo

r closing abdo

menNN

203122

79MHypert

ension,periph

eral vascular

disease, stenos

is art. mesente

ricaGastro

-enteric ische

mia, AMS endo

vascular treatm

entHemicolect

omy, Re-re-re

-laparotomy fo

r closing abdo

menYY

182723

75MHypert

ension, PTCA

Thoracic-abdo

minal aneurys

mRelapa

rotomy draina

ge abdomen

YY2622

2468F

Hypertension

, hypothyroid

,SCIAPThorac

ic-abdominal a

ortic aneurysm

sRelapa

rotomy for ste

nt replacemen

tNY

293025

55Mnone

Rupture of inf

rarenal aneur

ysm of aorta a

bdominalisRe

laparotomy fo

r abdominal co

mpartment syn

dromeNN

264026

22Mnone

Severe traum

aRelapa

rotomy forcec

umand colon

repairNN

282627

66FColitis

Ulcerosa

Severe traum

aRe-re-l

aparotomy fo

r removal Bog

ota baganddr

ainageNN

1928

55MHypert

ensionGastro

-enteric ische

mia after neph

rectomy

Relaparotomy

for resection

part of deode

num and jejun

umYNA

2823

Table 1b Contr

ol patients-de

mography, me

dical history,

reason admis

sion ICU and r

eason surgery

where biopsy

was obtained.

Nr

AgeGRemov

ed (Lung) Tum

orReleva

nt Medical His

toryMV(h)

BMIFEV1F

EV1 (%)FEV/ FVC %

FVCFVC (%)VC %

1*52M

pT3N0M0

(ex)Cigarette s

moker, DM II,

hypertension

1.524.62

.28680

.693.3076

NA02

*22M

pT1bN0M0

Cigarette smo

ker2.0

23.04.731

030.716

.66121NA

366M

pT2aN1M0

(ex)Cigarette s

moker, hyper

tension, prosta

te cancer, COP

D2.0

24.22.748

20.664

.17NA97

458F

pT1aN0M0

None2.0

28.42.851

040.773

.71NA11

65

60MpT1aN

1M0(ex)Cig

arette smoker

, lobectomy fo

r T1N0M0 lun

g cancer, pulm

onarytuberc

ulosis s/p succ

essful drug the

rapy1.5

24.43.809

70.675

.64113108

656M

pT4N2M0

(ex)Cigarette s

moker, COPD

2.021.23

.09780

.525.9111

8NA7

70MpT1bN

1M0Cigaret

te smoker, s/p

basal cell canc

er skin1.5

30.32.928

90.694

.25NA96

860F

pT2aN0R0

Hypertension

0.7526.32

.931270

.863.40N

A120

959F

CystCigaret

te smoker

0.7528.31

.97790

.75NANA

NA10

64FpT1bN

0M0Cigaret

te smoker, ch

olecystectomy

1.2526.01

.94760

.623.11N

ANA11

59FAdenoc

arcinoma CT2B

N2M1ACigaret

te smoker

0.7521.02

.09760

.643.2410

19412

52MpT2N0

M0Cigaret

te smoker

1.0022.53

.18810

.734.3611

0NA13

64MBronch

iectasisand inf

lammation,

benignHypert

ension, 6yr ag

o MI with PCI

of LAD artery

, DM II1.00

31.42.419

20.723

.348686

1474F

Inflammation

with central

necrosis,

benignBronch

iectasis and al

lergic broncho

pulmonary as

pergillosis

2.5021.31

.79820

.672.6910

3NA15

74MChondr

osarcoma

Colon cancer,

alcohol abuse

123.1

NA16

75MInflam

mation, benig

nTung b

ase tumor, T2N

2C0.5

25.63.049

90.594

.91121123

1756F

T1bN0Cigaret

te smoker, all

ergic asthma,

recurrent pne

umonia1

27.02.431

020.753

.24115111

1835M

Hamartoma, b

enignCigaret

te smoker

0.526.43

.971040

.864.6410

29819

40MpT1aN

0R0None

1.523.74

.941040

.736.7811

611320

73MpT2N0

(ex)Cigarette s

moker, atrial

fibrillation, no

n small cellul

ar lung carcin

omacT3TN

0M01.5

27.82.467

90.604

.0799NA

2168F

pT2N2(ex)Cig

arette smoker

, hypertension

, DM II0.75

29.42.41

030.822

.91104NA

2267M

Metastasis cle

ar cell renal c

arcinomaHyper

tension, DMII,

AVNRT, renal

cell carcinoma

, prostate canc

er1.25

26.7NA

2351M

T1aN0Cigaret

te smoker

1.2524.43

.56840

.65.9811

0NA24

58FpT0N0

Cigarette smo

ker, bronchiti

s2.5

22.8273

0.753.079

5NA25

61MpT1aN

0(ex)Cig

arette smoker

, seminoma of

testis2

33.22.416

40.584

.1686NA

2669M

pT3N1(ex)Cig

arette smoker

, hypertension

, iliofemoral by

pass1.5

32.52.738

60.763

.5887NA

2758M

pT3N1(ex)Cig

arette smoker

, hypertension

2.428.1N

ANAN

ANANA

NA

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Figure 1.Abnormal mitochondrial complexity of internal structure is denoted by black arrows,normal structure by black dashed arrows. Large aggregates of mitochondria are denoted by whitearrows, normal aggregates by dashed arrows. (A) Critically ill patient with many aggregates ofmitochondria that show some hyper internal complexity. (B) Control patient where mitochondriaappear normal with respect to number, size, shape, placement and internal architecture.Table 2. Electron Microscopy analyses# General Aggre-gates Number Position InternalComplexity General Aggre-gates Number Position InternalComplexityControl Critically Ill13 0 0 0 0 0 19 - 0 + 0 014 0 0 0 0 - 20 0 + 0 0 015 0 + 0 0 0 21 -- + + -- 016 - 0 + 0 - 22 -- 0 + -- +17 -- 0 0 0 - 23 -- 0 0 0 -18 0 0 0 0 0 24 - + 0 0 024 0 + 0 0 - 25 - + 0 - 0Legend Table 2. General impression/Placement: -- moderately abnormal, - mildly abnormal, 0normal. Symbols for Number of aggregates/number of mitochondria/ size: 0 normal,+higher/large. Internal complexity:- hypo complex, 0 normal, + hyper complex.

Mitochondrial dynamicsSince PGC1-α is regarded as the master regulator of mitochondrial biogenesis, wemeasured its protein content. Compared to controls, PGC1-α was significantly lower incritically ill patients (0.084±0.004 vs. 0.064±0.006 A.U.) (-24±7%), p=0.016 (Figure 2)and phosphorylated AMPK relative to total AMPK (pAMPK/AMPK), an indicator ofmetabolic stress, was higher in critically ill patients (control vs critically ill 1.02±0.04vs. 1.66±0.14 (+63±14%) p=0.001).Content of proteins related to mitochondrial fusion were significantly lower in criticallyill patients: control vs critically ill, respectively, Mfn1 0.48±0.03 vs 0.40±0.02 (-16±4%,p=0.046), Mfn2 0.076±0.003 vs. 0.066±0.001 (-13±2%, p=0.008) and OPA10.082±0.003 vs. 0.073±0.001 (-11±2%, p=0.010). The content of fission protein DRP1was significantly higher, control vs critically ill: 0.48±0.03 vs. 0.58±0.04 (+21±5%,

IMPAIRED MITOCHONDRIAL DYNAMICS AND DECREASED COMPLEXES IN DIAPHRAGM FIBERS OFCRITICALLY ILL PATIENTS

94

p=0.04), see Figure 3. These results suggest that in critically ill patients, mitochondrialdynamics are shifted towards more mitochondrial fission, and hence mitochondrialfragmentation.

Figure 2.Representative example of Western blot with antibodies for PGC-1α and pAMPK/AMPK. Incritically ill patients the protein levels of PGC-1α were significantly lower and the ratio ofpAMPK/AMPK was significantly higher.

Figure 3. Representative example of Western blot with antibodies for fusion protein Mfn1, Mfn2,OPA1 and fission protein DRP1. Fusion proteins were significantly lower and fission proteinsignificantly higher in critically ill compared to controls (*= p<0.05).

Mitochondrial complexesContentThe sum of the staining intensity of all mitochondrial complexes was significantly lowerin critically ill patients. Control vs critically ill respectively 1.4±0.10 vs. 1.0±0.11 (-31±7.6%, p=0.005). Complex III (0.58±0.05 vs. 0.39±0.05; -35±8.5%, p=0.014),complex IV (0.22±0.02 vs. 0.15±0.02; -38±9.2%, p=0.014) and complex V (0.37±0.03 vs.0.28± 0.03; -27±7.9%, p=0.028) were all significantly lower in critically ill patientscompared to controls. Content of mitochondrial complex I (0.11± 0.01 vs. 0.08 ±0,02; -25±13.3%, p=0.15) and II (0.12±0.02 vs. 0.10±0.01; -13±12.8%, p=0.19) was not

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significantly lower in critically ill patients, see Figure 4 (for clarity control data werenormalized in this figure to 100%).

Figure 4. (A) Representative example of Western blot with mito profile antibody cocktail. Lane 1 and2 are diaphragm homogenates of critically ill patients, lane 3 rat heart loading control and lane 4and 5 diaphragm homogenates of control patients. (B) Content of mitochondrial complexes ( *=p<0.05).

ActivityWe measured the activity of succinate dehydrogenase (SDH) in diaphragm cryosectionsper fiber type (Figure 5A shows representative examples). We did not find significantdifferences in SDH-activity controls vs critically ill in slow-twitch fibers (0.14 ± 0.01A660 µm-1 s-1 vs. 0.15 ± 0.02 A660 µm-1s-1. p=0.57) or in fast-twitch fibers (0.12 ± 0.01 A660µm-1s-1 vs 0.12 ± 0.01 A660 µm-1s-1, p=0.86) (Figure 5B), which is in line with our findingsof unaffected protein levels of complex II.

Figure 5. (A) Images fast-MyHC (top, grey surfaces) and of SDH-activity (bottom) on cryosections ofa representative control (left) and a representative critical ill patient (right). Symbols indicatecorresponding fibers, filled symbol: fast-twitch, empty symbol: slow-twitch. (B) Mean activity ofcomplex II on the sections did not differ between control and critically ill patients in slow- nor fast-twitch fibers.

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Fiber type distributionSince mitochondrial density is higher in slow-twitch compared to fast-twitch musclefibers, we verified whether the reduced content of complexes of the electron transportchain or preserved SDH activity was a reflection of a fiber type switch.Immunohistochemistry analyses did not show a difference in fiber type fraction(slow/fast-twitch) between control and critically ill patients, which was respectively51%/49% vs. 53%/47% (Figure 6A, p=0.59). The fiber area fraction of slow/fast-twitch fibers was respectively 52%/48% and 56%/44% p=0.24 (Figure 6B). Thus,decrease of mitochondrial protein content or perseverance of SDH activity in criticallyill patients was not caused by a fiber-type shift.

Figure 6. (A) Fraction of fibers typed slow/fast-twitch and (B) the fraction of cross sectional areacovered by slow/fast-twitch fibers did not differ between control and critically ill patients.

Mitochondrial respirationIn order to understand the functional consequences of the alterations in mitochondrialstructure and dynamics, we performed mitochondrial respirometry in a subgroup ofpatients. Figure 7A shows a typical example of the respirometry recordings.

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Figure 7. A) Typical example of an experimental recording of mitochondrial respiration (criticallyill patient #27). Upper panel shows oxygen concentration (grey line), lower panel oxygen flux permass (black line). First, complex I substrates glutamate (G), malate (M) and pyruvate (P) areinjected into the measuring chamber and respiration is supported by proton leak. Subsequently, ADP(A) is added, that supports complex I-coupled respiration. Cytochrome C (C) is added to testmembrane integrity. The addition of succinate (S) provides a combined complex I- and II-coupledrespiration, also called maximal oxidative phosphorylation. Subsequently FCCP (F) is titrated thatuncouples respiration, enabling determination of maximal respiratory electron transfer capacity.Rotenone (R) blocks complex I function and the respiration observed is supported by complex II-uncoupled respiration only. B) Mean respiratory values for each of the conditions given in (A) perpatient group (control: grey, critically ill: black).

Leak respiration did not differ between controls and critically ill patients (8.5 ±1.4 vs.5.9 ±1.6 pmol s-1 mg-1 respectively; p=0.23). Maximal complex I coupled-respirationdid not differ between controls and critically ill patients (20.9±3.9 vs. 18.6±5.3 pmol s-1 mg-1, respectively p=0.74). None of the samples showed an increase in respiratory rate(>15%) after addition of cytochrome c, indicating an intact mitochondrial outer-membrane. Maximal oxidative phosphorylation, by maximal coupled complex I- and II-coupled respiration, did not differ between controls and critically ill patients (41.1 ± 6.5vs. 40.5±12.1 O2 pmol s-1 mg-1 respectively, p=0.97). Uncoupling mitochondrialrespiration (complex I to IV) from ATP production (complex V and transporters) didnot alter these results (p=0.94). Maximal complex II-uncoupled respiration did notdiffer between controls and critically ill patients (28.0± 3.3 vs. 29.8± 7.8 pmol O2 s-1 mg-1, respectively; p=0.81). No differences were observed in maximal complex I respirationnormalized to maximal oxidative phosphorylation (p=0.41) or the ratio of complex Irespiration versus leak respiration, named complex I respiratory control ratios(p=0.70) (data not shown).Heterogeneity in mitochondrial function in critically ill patientsMean mitochondrial respiration in diaphragm samples of critically ill patients did notdiffer from controls. However, we did notice a large heterogeneity in respiration in thecritically ill patients (Figure 8 and Table 4). Some critically ill patients had remarkablylow maximal mitochondrial respiratory capacity and these same patients all had low

IMPAIRED MITOCHONDRIAL DYNAMICS AND DECREASED COMPLEXES IN DIAPHRAGM FIBERS OFCRITICALLY ILL PATIENTS

98

protein content of mitochondrial complexes (y=17.99+0.274x, R2=0.264, p=0.0246).These data may indicate that some critically ill patients were more susceptible tomitochondrial damage compared to other patients.Table 4. Heterogeneity mitochondrial function

Figure 8. (A) Western blot images of mitochondrial complexes for critically ill patient lane 1 control,lane 2-4 respectively critically ill patients #17, #18 and #22, lane 5 rat heart loading control. Notethat content of mitochondrial complexes decreased in the three critically ill patients. (B) Signalrecording of mitochondrial respiration: #17 had almost no respiration, and respiration of #22 wasvery low.

Discussion

This study shows that in diaphragm fibers of critically ill patients (1) the levels ofmitochondrial fission proteins are increased and the level of mitochondrial fusionproteins are decreased; (2) the levels of PGC1-α are decreased; (3) activation of the

Patient Mitochondrialprotein content Max OXPHOS(pmol s-1 mg-1) Max Oxidative capacity(pmol s-1 mg-1)17 0.33 0 018 0.56 1.0 1.722 0.23 18.1 17.0Mean±SD Critically Ill 1.0±0.11 40.47±12.09 44.52±11.58Mean±SD Control 1.4±0.10 41.07± 6.51 43.69 ±4.40

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energy stress sensor AMPK is increased; (4) the content of electron transfer proteincomplexes III, IV and V are decreased, and that (5) maximal mitochondrial respirationis unaffected.Mitochondrial dynamics and ROSSince fission and fusion are physiological processes that are essential for maintainingmitochondrial quality, disturbed mitochondrial fragmentation contributes tomitochondrial dysfunction. Recent work showed disrupted mitochondrial function andbiogenesis in diaphragm specimens of mechanically ventilated brain dead organ donors73. The present findings indicate that in critically ill patients the mitochondria undergoincreased fission and decreased fusion. Disturbed fission and fusion may affectmitochondrial networks, limit efficient mixing of the matrix and inner membrane andoptimal ATP production 138. If the mitochondria fail to produce ATP during reloading ofthe diaphragm when respiratory support is decreased or discontinued, this may impairweaning from mechanical ventilation or even prolong time on the ventilator in criticallyill patients.Mitochondrial proteins and mtDNA in fragmented mitochondria are very susceptible todamage by mitochondrial reactive oxygen species 133,138. The mitochondria are animportant source of ROS; Although the underlying mechanisms are incompletelyunderstood, increased ROS may result from mitochondrial fission 142,143, disturbedcalcium handling (Ingalls et al., 2015), increased levels of mitochondrial fatty acidhydroperoxides 144, and impaired protein import into mitochondria 145. Unloading ofthe diaphragm, as occurs during controlled mechanical ventilation, has been suggestedto result in a state of metabolic oversupply, as indicated by large deposits of fat andglycogen 73. Metabolic oversupply has been suggested to inhibit the electron flow, whichreduces ATP synthesis. This may lead to increased leakage of electrons from theelectron transport chain, and hence forming reactive oxygen species 73. ROS maydirectly - or indirectly via mitochondrial damage - induce activation of AMPK and otheratrophic pathways 35,36, that both are activated in the diaphragms of critically ill patients(Figure 2 and Hooijman et al 130). Therefore, it would be of interest to investigateoxidative damage in the diaphragm of these patients, for instance via Oxyblots andanalysis of nitrosylation.Another pathway via which mitochondria are involved in regulating muscle atrophy isvia the release of pro-apoptotic factors 37. When mitochondrial outer membranes aredamaged, cytochrome c can leak from the mitochondria into the cytosol and thisinitiates apoptosis. Thus, mitochondria may play a role in regulating muscle atrophyand mitochondrial dysfunction may impair diaphragm function, which is especiallyimportant during weaning from mechanical ventilation in critically ill patients.

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Mitochondrial respirationMitochondrial respiration was not impaired in a subgroup of patients. Earlier findingsalso showed that complex I and II-stimulated respiration in mitochondria isolated fromdiaphragm muscles of 12h 140 and 49h 41 mechanically ventilated animals waspreserved, while total mitochondrial protein content was decreased. Our finding thatthe average critically ill patient had unaffected complex I protein content is in line withthe comparable maximal complex I respiration between groups. Similarly, the absenceof a difference in protein content and activity of complex II may be in accordance withthe unaffected complex II-coupled respiration in diaphragm samples of critically illpatients. Nonetheless, the protein content of complex III, IV and V were significantlyreduced in the critically ill patients. Our observations of high protein content of complexIV and V compared to other complexes (in both patient groups) are also in accordancewith earlier observations of their excess capacity 146. Our findings of decreased complexIV content in critically ill patients compared to controls suggest that this excess capacityis reduced in mechanically ventilated critically ill patients. This may have functionalconsequences for mitochondrial respiration at submaximal level and in conditions oflow oxygen availability 146. The molecular cause is unknown, but might relate todifferent vulnerability and/or turnover rate of these proteins 147.Leak respiration did not differ between critically ill patients compared to controlsAlthough slightly simplified, the leak state can be considered as an indication of protonleaking through the inner membrane where protons that are pumped by the electrontransport chain leak back into the matrix. Via this mechanism, the production of ATP bycomplex V is bypassed, resulting in heat production as a protective mechanism toreduce the production of mitochondrial superoxide 148. To the best of our knowledge,the only study on mitochondrial respiration in human diaphragm is from Martin et al.They observed that intermittent phrenic nerve stimulation increased leak respirationof the stimulated hemi-diaphragm compared to the unstimulated hemi-diaphragm 149.These findings indicate that leak respiration may rapidly change in response to externalstimuli. Whether mitochondria of mechanically ventilated critically ill patients useother mechanisms to prevent production of super oxide, such as downscaling ATPsynthase content (Figure 4) or increased fission, should be the topic of futureexperiments.Sarcolemmal and intermyofibrillar mitochondriaSarcolemmal and intermyofibrillar mitochondria have different energetic,compositional and biochemical characteristics 147,150–152 and respond differently to(patho)physiologic stimuli such as hypoxia, disuse and disease and apoptopic stimuli37,153,154. Unfortunately, the methods applied in the current study do not allow todistinguish between these types of mitochondria..

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Clinical implicationsIn diaphragm samples of mechanically ventilated critically ill patients, we observedmitochondrial fragmentation and decreased content of mitochondrial complex III-V,which may contribute to weaning failure. The patient group studied here comprised of28 critically ill patients who are, with respect to age, duration of ICU stay and underlyingdisease, representative of critically ill patients that are admitted to the typical ICU. 155.However, for exploration of the relation between mitochondrial function and specificdisease entities, medication, age and gender, duration of MV, more expanded studiesare needed because of the variability among patients.Our current findings of increased activation of AMPK and reduction of PGC1-α givereason to focus on future treatment strategies that influence FoxO-3 dependentmechanisms to inhibit the ubiquitin-proteasome pathway, that is activated indiaphragms of critically ill patients 130. Recently, a number of drugs are identified thatinduce PGC1a mRNA, for example microtubule inhibitors 156, and that inhibit AMPK,such as compound C 157. Further experiments are required to test whether such drugsmay inhibit atrophic genes and may help to prevent mitochondrial diaphragmdysfunction.Since the diaphragms of critically ill patients experience low energetic demands, wehypothesize that their mitochondria undergo enhanced fission to help to maintainbioenergetics capacity and to eliminate damaged mitochondria by autophagy. When theenergy demands of the diaphragm muscle fibers rise during weaning from mechanicalventilation, the fragmented mitochondria may fail to produce ATP efficiently and thismay contribute to weaning failure.Methods

PatientsDiaphragm muscle biopsies were obtained from mechanically ventilated critically illpatients (n=28) in whom elective or emergency surgery was performed and frompatients undergoing resection of a suspected early lung malignancy (controls, n=27).Exclusion criteria were COPD (GOLD III/IV), chronic heart failure, neuromusculardisease, chronic metabolic disorders, pulmonary hypertension, chronic use ofcorticosteroids (>7.5 mg/day for at least 3 months before hospital admission), and>10% weight loss within the last 6 months. The biopsy protocol was approved by theinstitutional review board at the VU University Medical Center Amsterdam. Writteninformed consent was obtained from the patient or a legal representative.Biopsy handlingBiopsies of the diaphragm muscle were obtained during surgery and cut into smallersections. One part was directly frozen in liquid nitrogen and stored at -80°C forhistology experiments, analysis of enzymatic activity on cryosections and Western blot

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analysis. Another part of the biopsy was prepared for respiratory measurement andstored at 4°C in a preparation solution. Another part was fixated for electronmicroscopy in glutaraldehyde cacodylate buffer (for the composition of solutions anddetailed protocol see below). Not all measurements could be performed on all biopsiesdue to size limitations.Electron MicroscopyOne part of the fresh biopsy was fixed in 2.5% glutaraldehyde in 0.1M sodiumcacodylate buffer for electron microscopy, that was performed at the ElectronMicroscopy Core Facility at the Medical College of Wisconsin (USA). Specimens wereembedded in Epon resin using standard techniques and 1μm sections of the tissue werestained with toluidine blue to provide scout sections to evaluate tissue quality. Eponblocks containing tissue fragments of appropriate quality were sectioned at 30 nm andstained with 2% uranyl acetate and Reynold’s lead citrate. Electron micrographs weretaken using a Hitachi H600 transmission electron microscope at a range ofmagnifications (3500-30.000x) and assessed by a blinded neuro-pathologist forpertinent abnormalities.Western blottingMitochondrial dynamicsThe content of PGC1α, the ratio of the active (phosphorylated) and total form of AMPKand key proteins of fission ((dynamin-related protein-1 (DRP1)) and fusion (opticatrophy 1 (OPA1) and mitofusion 1 and 2 (Mfn1 and Mfn2)) was assessed by Westernblotting. The protocol for Western blotting was similar as described previously 34.In brief, muscle samples that were directly after obtainment frozen in liquid nitrogenand stored at -80°C, were pulverized and immediately re-suspended in a lysis buffer(20 mM Tris-HCl, 1% Triton X100, 10% glycerol, 150 mM NaCl, 5 mM EDTA, 100 mMNaF and 2 mM NaPPi supplemented with protease inhibitors (1:50, Sigma-Aldrich) andphosphatase inhibitors (1:100, Sigma-Aldrich) and 1 mM PMSF. The homogenateobtained was centrifuged at 18000 g for 20 min at 4°C and the supernatant stored at−80°C until use. Because of size limitations, samples of 4 biopsies from the same groupwere pooled with equal protein quantity from each sample. Equal amounts of muscleprotein were loaded on pre-cast gels (Bio-Rad, Any kD; Hercules, CA, USA). Proteinswere electro-transferred to PVDF membranes at 35 mA overnight. The membraneswere probed with specific primary antibodies anti-rabbit α tubulin (loading control;Sigma-Aldrich), anti-mouse Mfn1 (Abcam), anti-rabbit Mfn2 (Abcam), anti-rabbit OPA1(Abcam), anti-rabbit DRP1 (Cell Signaling, Danvers, MA, USA), anti-rabbit PGC-1α(Abcam), anti-rabbit p-AMPK (thr 172, Cell Signaling), anti-rabbit AMPK (CellSignaling). Thereafter, the membranes were incubated with appropriate HRP-conjugated secondary antibodies. Protein bands were visualized by an enhanced

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chemiluminescence method and protein content was assessed by determining thebrightness–area product 158.Mitochondrial complexesThe content of mitochondrial complexes I-V in diaphragm muscles were analyzed byWestern blotting. Muscle samples were freeze dried and after weighing homogenizedin electrophoresis buffer containing 62.5 mM Tris-HCl(pH 6.8), 15% glycerol, 2% SDS,0.0025% brome phenol blue, 40 mM DTT and protease and phosphatase inhibitorcocktails (Sigma P8340, P5726, P0044-100x diluted in the buffer). Proteinconcentration of the homogenates was determined with the Pierce 660nm proteinassay and subsequently adapted with buffer to a concentration of 2.5 µg/µl. On 18 well8-16% TGX gradient gels 10 μg diaphragm homogenates were run in duplo, togetherwith 7.5 µg protein of rat heart reference homogenate. The gels are blotted in a semidryblotting apparatus on PVDF membranes, the blots are blocked overnight in a 5% milksolution in TBS-T. The next day the blots are incubated for one hour in a 1000x dilutionof mito profile antibody cocktail (Abcam mitoProfile total OXPHOS Rodent WB antibodycocktail AB110413) and subsequently washed three times with TBS-T. Then the blotsare incubated for one hour in a 5000x dilution of goat anti mouse horse radishperoxidase in TBS-T and again washed three times with TBS-T. Finally, blots werestained with enhanced chemiluminescence (ECL) prime reagens. Blots were scannedwith LAS 3000. For quantifiable chemiluminescence imaging a LAS 3000 detectionsystem and analysis software were used for band quantifications. Rat hearthomogenate was used as loading control and for normalization of protein levels.Histology of mitochondrial functionSuccinate dehydrogenase activitySerial 10-µm cross sections from the diaphragm muscle were cut in a cryostat at -20°Cand placed on polylysine-coated slides. The determination of succinate dehydrogenase(SDH) activity was similar as described before 159. In brief, sections were air-dried for30 min at room temperature and incubated for 20 min at 37°C in a medium consistingof 0.4 mM tetranitroblue tetrazolium (Sigma, St Louis, MI, USA), 75 mM sodiumsuccinate, 5mM sodium azide, and 3.5 mM sodium phosphate buffer (pH 7.6). Thereaction was subsequently stopped in 10 mM HCl, and sections were washed andembedded in glycerin/gelatin. Photos were taken using an interference filter at 660 nmon microscope (Leica, Wetzlar, Germany) and calibrated with grey filters. Absorbancemeasurements were made. Images were obtained with a 20x objective and amonochrome charge-coupled device camera (Sony XC77CE, Towada, Japan) connectedto a LG-3 frame grabber (Scion, Frederick, MD, USA) in a Power Macintosh computer(Apple Computer Benelux BV, Bannik, The Netherlands). Images were analyzed usingNIH ImageJ (NIH, Bethesda, USA). Per biopsy, around 20 randomly selected musclefibers were analyzed and subsequently averaged. SDH activity was calculated as the

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increase of absorbance at 660 nm per μm section thickness per second of incubationtime (ΔA660 μm-1s-1).Since mitochondrial volume density is different between fast- and slow-twitch musclefibers 160, we determined SDH activity per fiber type using MyHCimmunohistochemistry. We determined myosin heavy chain isoform composition inserial sections, as previously described 72 using immuno-fluorescent staining of fast-myosin heavy chain. Serial 10 µm sections were rehydrated for 10 min in phosphatebuffer (PBS) and subsequently blocked with PBS containing 0.5% (wt/vol) bovineserum albumin (PBS-0.5%BSA, Molecular Probes). Subsequently, sections wereincubated with a primary antibody against fast MyHC (1:50 MY-32, Abcam) followed byincubation with a fluorescent labeled secondary antibody (Alexa fluor 647, MolecularProbes, Eugene, OR). Finally, individual muscle fibers were visualized by fluorescentWGA (1:25 diluted in PBS-0.5%BSA, Molecular Probes) which selectively recognizessialic acid and N-acetylglycosaminyl sugar residues. Between stainings, sections werewashed 3 times for 3 min with PBS. Samples were washed and protected with a layerof Vector Shield and a cover glass. Sections were analyzed with use of an inverted digitalimaging microscopy workstation [Intelligent Imaging Innovations (3i)] equipped witha motorized stage and multiple fluorescent channels. A cooled charge-coupled devicecamera (Cooke Sensicam; Cooke, Eugene, OR) was used to record images. Exposures,objective, montage, and pixel binning were automatically recorded. Dedicated imagingand analysis software (SlideBook, version 4.2, 3i) was obtained from IntelligentImaging Innovations (Denver, CO). Cross sectional areas were calculated by ImageJsoftware.Mitochondrial respirometryMitochondrial respiration was determined as described previously 160,161 with smallmodifications. Fresh diaphragm biopsy parts (~15mg) were manually dissected intostrips, not disrupting fiber structure and permeabilized for 30 min at 4 °C in 50 µg mL-1 saponin in preparation solution containing 2.8 mM CaK2EGTA,7.2 mM K2EGTA, 5.8 mMATP, 6.6 mM MgCl2,20 mM taurine, 15 mM phosphocreatine, 20 mM imidazole, 0.5 mMDTT and 50 mM MES; pH 7.1, adjusted with KOH. Tissue was subsequently washed inmitochondrial respiration solution, containing 0.5mM EGTA, 3mM MgCl2, 60mM K-lactobionate, 20mM taurine, 10mM KH2PO4, 20mM HEPES, 110mM sucrose and 1 g L-1fatty acid free BSA (pH 7.1), quickly blotted dry, weighed and transferred to arespirometer (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). Oxygenconcentration was maintained above 300 μM throughout the measurements to avoidlimitations in oxygen supply. Maximal complex I-coupled respiration was measured inMiR05 after addition of 10 mM sodium glutamate, 2 mM sodium malate and 5 mMsodium pyruvate and 2.5 mM ADP. Outer-mitochondrial membrane integrity was tested

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by addition of 10 μM cytochrome c. Maximal oxidative phosphorylation, withsimultaneous input of electrons through complex I and II, was measured after additionof 10 mM succinate. Subsequently, un-coupler carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) was titrated to assess maximal electron transportchain activity. Subsequently, complex II uncoupled respiration was measured afterblocking complex I by adding 0.5 μM rotenone. Residual oxygen consumption,measured in the presence of 100mM sodium azide, was subtracted from all values.Measurements were performed at 37°C. Two tissue samples from the same diaphragmbiopsy were measured simultaneously and the data was averaged. Values are expressedas oxygen flux per time per wet weight muscle tissue (pmol s-1 mg-1).Statistical analysisDue to the limited biopsy size not all parameters were determined in all biopsies (seeTable 2). All data is represented as the mean ± standard error of mean (SEM). Outcomeof continuous measurements that were normally distributed were compared usinggroup t-tests, outcome of continuous measurements that were not normally distributedwere compared using Mann-Whitney tests (Real Statistics Resource Pack software3.2.1). Fisher’s exact test (2-tail) was used to compare critically ill and control groupswith respect to categorical data using GraphPad Prism version 6.00 for Windows(GraphPad Software, La Jolla California, USA). Differences between groups wereattributed to chance unless they were significant at the 0.05 level.

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