Muscle contractility of leg muscles in patients with mitochondrial myopathies

Accepted Manuscript Muscle contractility of leg muscles in patients with mitochondrial myopathies

Nanna Scharff Poulsen, Julia Rebecka Dahlqvist, Gitte Hedermann, Nicoline Løkken, John Vissing PII: DOI: Reference:

S1567-7249(17)30341-0 doi:10.1016/j.mito.2018.07.001 MITOCH 1298

To appear in:

Mitochondrion

Received date: Revised date: Accepted date:

19 December 2017 24 May 2018 5 July 2018

Please cite this article as: Nanna Scharff Poulsen, Julia Rebecka Dahlqvist, Gitte Hedermann, Nicoline Løkken, John Vissing , Muscle contractility of leg muscles in patients with mitochondrial myopathies. Mitoch (2018), doi:10.1016/j.mito.2018.07.001

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Muscle contractility of leg muscles in patients with mitochondrial myopathies

Nanna Scharff Poulsen1 , BSc, Julia Rebecka Dahlqvist1 , MD, Gitte Hedermann1 , MD, Nicoline

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Løkken1 , MD, and John Vissing1 , MD, DMSc

Copenhagen Neuromuscular Center, Rigshospitalet, Copenhagen University Hospital, 2100

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Copenhagen, Denmark.

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Correspondence to: Nanna Scharff Poulsen

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Copenhagen Neuromuscular Center, section 6921 Rigshospitalet

Phone: +45 35457357

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DK-2100 Copenhagen, Denmark

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Juliane Maries Vej 28

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E-mail: [email protected]

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Abstract Background The primary disease mechanism underlying mitochondrial myopathies (MM) is impaired energy generation to support muscle endurance. Little is known about muscle contractility before energy becomes deficient during muscle contractions. We investigated muscle contractility

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in MM to uncover potentially fixed weakness aspects of the disorders.

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Methods Contractility of calf and thigh muscles was investigated by comparing strength with

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contractile cross-sectional area (CCSA) of the used muscles, as measured by stationary

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dynamometry and MRI, respectively.

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Results and discussion Our findings suggest reduced contractile properties in thigh and calf

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muscles of patients with MM.

Abbreviations: CCSA: contractile cross-sectional area, CK: creatine kinase, CPEO: chronic progressive external

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ophthalmoplegia, CSA: cross sectional area, CypD: cyclophilin D, MD: mitochondrial disease, MM: mitochondrial

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myopathy, PT: peak torque, ROI: region of interest

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1 INTRODUCTION Mitochondrial diseases (MD) are caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) leading to impaired function of the respiratory chain and reduced energy generation (1). Symptoms in patients with MD usually first arise in highly oxidative tissues such as

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the CNS, skeletal and cardiac muscles and endocrine glands (2). Syndromes that mainly affect

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skeletal muscles are called mitochondrial myopathies (MM).

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In patients with mtDNA mutations, tissues are differentially affected according to the oxidative requirement and mutation type and load of the tissue (3,4). The threshold at which skeletal muscle

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symptoms arise varies with the type of mutation. For the common mutation, m.3243A>G, a

deletions, the threshold is 50-60 % (5).

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mutation load of 70-80 % is required before muscle symptoms emerge (5). For single, large-scale

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A hallmark of healthy muscles is the linear relation between muscle strength and muscle crosssectional area (CSA) (6,7), often used as an indicator of muscle quality (8). This relationship, which

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depicts muscle contractility, is disrupted in some muscle diseases (6). In patients with MM, muscle function is generally believed to be affected primarily by impaired oxidative phosphorylation,

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which leads to low aerobic fitness and exercise intolerance. However, there is also some evidence for a more static and structural affection of the muscles in MM, which can involve fatty replacement of muscle (9,10), transformation of type I fibres to type II fibres (11), decreased mitochondrial quantity and quality (11) and increased expression of cyclophilin D (a modulator of the mitochondrial permeability transition pore) (12). Such changes are not universal for all MMs, and may depend on the genotype and the mutation load (9,10). Evidence for a more fixed muscle affection has also been drawn from a mouse model of MM, showing that the specific force (force per CSA) is decreased in muscle fibres (12).

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Fatigue and decreased muscle contractility caused by energy deficiency during sustained exercise is well recognized in MM, but it is unknown whether fixed weakness is an issue as well in these disorders(13). We therefore wanted to shed light on this question by investigating the relation between muscle peak torque (PT) and contractile cross-sectional area (CCSA) in muscles of

AND METHODS

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2 MATERIALS

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patients with MM using static dynamometry and MRI.

The study was approved by the Danish Data Protection Agency and the local ethics committee

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(study no.: H-16000588), and was conducted in accordance with the declaration of Helsinki. All

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subjects gave written informed consent to participate.

2.1 STUDY DESIGN

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The study is observational and cross-sectional by design. Subjects were examined by Dixon MRI to calculate muscle fat fraction, CSA and CCSA (calculated by subtracting the amount of fat from the

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CSA) of muscles in calf and thigh, and by stationary dynamometry to calculate the maximal muscle strength of the scanned muscle groups. Through these measures, we could investigate the

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contractility as the relation between muscle PT and muscle CCSA. Inclusion criteria were: 1) genetically verified MM and 2) age more than 18 years old. Exclusion criteria were: 1) competing disorder that could interfere with the results (i.e. arthritis or other muscular disorders) and 2) contraindications for MRI.

2.2 SUBJECTS All patients were recruited from the Copenhagen Neuromuscular Center, Rigshospitalet.

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18 women and 17 men with MM (age 47 years range [19-76], BMI 24 kg/m2 [15-38]) were compared to 18 healthy women and 20 healthy men (age 47 years [25-76], BMI 24 kg/m2 [20-31]).

Patients with MM were divided into subgroups based on their genetic defect (Table 1). The two

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main subgroups were patients carrying single, large-scale deletions in mtDNA (n=17) and the m.3243A>G point mutation (n=12). The remaining 6 patients carried private mutations and had a

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heterogenous clinical presentation (Table 1). All patients with single, large-scale deletions had

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chronic progressive external ophthalmoplegia (CPEO). None with other mutations had CPEO. The contractility was further investigated in patients with high (n=15) and low (n=12) mutation load

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in skeletal muscle.

Some of the MRI and strength data from the healthy controls and CPEO patients have been

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published in a different context (10,14,15).

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Table 1: Patient characteristics BMI Mutation (kg/m² )

Mutatio Clinical presentation n load in skeletal muscle (%)

23

m.3243A>G

ND

FSGS, HI, EI, ASD, asthma

2

62

M

27

m.3243A>G

ND

76

3

43

M

20

m.3243A>G

ND

Polio as a child, right-side paresis MIDD, EI

4

37

F

23

m.3243A>G

ND

MIDD, EI, asthma

71

5

41

F

18

m.3243A>G

ND

MIDD, EI

116

6

71

F

31

m.3243A>G

50

EI, dysphagia

62

7

52

F

26

m.3243A>G

2

8

50

F

27

m.3243A>G

67

9

49

M

26

m.3243A>G

10

61

M

25

m.3243A>G

11

58

F

22

m.3243A>G

12

41

M

30

13

19

M

14

23

15

Creatin e kinase, U/L

92

No clinical symptoms EI

119

No clinical symptoms

34

DM, EI

27

SLE

489

m.3243A>G

67

EI

111

17

m.8,483_13,459del

95

CPEO, EI, AV-conduction block 146

F

25

m.9,412_14,410del

83

CPEO, EI

23

M

20

m.8,937_13,030del

63

CPEO, DM

176

16

36

M

22

m.6,694_12,988del

33

CPEO, EI

129

17

42

M

24

m.8,483_13,459del

58

CPEO, EI

18

45

M

21

70

CPEO, EI, dysphagia

162

19

50

M

27

m.13,585_15,596de l m.7,177_13,767del

51

CPEO, EI

742

20

52

F

32

ND

CPEO, EI, migraine, dysphagia

130

21

52

F

19

m.11,717_15,704de l m.6,098_12,201del

CPEO, EI

201

22

60

F

20

77

CPEO, MS, EI, dysphagia

340

23

66

F

23

m.12,113_14,422de l m.9,161_12,965del

69

CPEO, EI, tremor

146

24

66

M

23

m.8,483_13,460del

35

CPEO

188

25

67

F

26

m.9,110_14,605del

63

CPEO, EI, HI, dysphagia

369

26

69

M

15

36

CPEO, dysphagia, PEG, EI, GR

74

27

76

F

22

m.12,113_14,421de l m.12,113_14,421de

ND

CPEO, EI

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Subjec Ag Sex t no. e (M: (y) Male, F: Female ) 1 36 M

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l 36

M

23

100 bp deletion

50

CPEO

29

40

M

24

m.12,294G>A

75

CPEO

1290

30

19

F

19

m.4,450G>A

67

EI

<200

31

50

F

18

m.4409T>C

73

EI, fatigue

267

32

43

M

28

m.5,132del AA

90

EI

125

33

50

F

25

m.4,087A>G

100

EI, myoglobinuria

953

34

53

F

21

m.8,344A>G

99

EI, lipoma

824

35

24

F

38

m.8,344A>G

78

EI, Lipoma, encephalopathy

238

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There is no other clinical feature of CNS or PNS lesions than described.

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ASD = atrium septum defect; BMI = Body Mass Index; CPEO = Chronic Progressive External Ophthalmoplegia; DM = Diabetes Mellitus; FSGS = focal segmental glomerulosclerosis; EI = Exercise Intolerance, GR = Growth Retardation; HI = Hearing

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Impairment; MIDD = maternal inherited diabetes and deafness; MS = Multiple Sclerosis; ND = Not Determined; PEG =

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percutaneous endoscopic gastrostomy; SLE = systemic lupus erythematosus.

2.3 MUSCLE STRENGTH MEASUREMENTS

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Isokinetic muscle strength (highest peak torque in Newton meter, Nm) of knee flexors and extensors and ankle dorsal and plantar flexors were tested using a Biodex System 4 PRO

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dynamometer (Biodex Medical Systems, NY, computer software program version 4X) as described

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before (6,10). Velocities of 90o s-1 and 45o s-1 were used for knee and ankle analysis respectively.

Only results from the right leg are shown, since limb strength was measured to be symmetric in a subgroup of the patients. One patient, however, had right-sided paresis after polio as a child, why his left leg was tested.

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2.4 MRI MRI was used to calculate the CSA and fat content of muscles in thigh and calf, thereby making it possible to calculate the CCSA. The relation between PT/CCSA and fat content was further investigated. MRI settings

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2.4.1

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Subjects were scanned in a head first, supine position in a 3.0 T Siemens scanner (Magnetom Verio Tim System; Siemens AG, Erlangen, Germany) using a spine coil, body matrix and peripheral

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angio coil. The MRI protocol included three-planned localizers, axial T1-weigthed imaging for

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qualitative analysis of muscle and axial Dixon imaging for quantitative measurements of fat

reviewed by the same investigator (NSP).

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involvement in muscles, using technical specifications as previously described (14). All scans were

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All scans were performed using the length of the femur and tibia bones as reference.

All data were analysed on a Siemens Syngo MR Workplace using Numaris/4 B17 software. Former

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studies have shown that estimations of CSA from a single MRI slice can be representative of a whole muscle (16,17). The best correlation between CSA and muscle volume (MV) is found close

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to the maximal CSA (16), which is why we used the slice with the largest CSA as our region of interest (ROI) (see Figure 1 for further explanation). 2.4.2

Analysis of thigh

The localizer was used to measure the length of femur. By adding slice positions from the Dixon scan, the ROI could be found, and the muscles could be manually outlined. Since the size of the CSA of the anterior (quadriceps femoris and sartorius) and posterior muscle groups (semitendinosus, semimembranosus and biceps femoris) differ along the length of femur, we conducted a small study to find the ROI. The CSA was estimated at 30 %, 35 %, 40 %, 50 %, 60 %,

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65% and 70 % of the length of femur (prox-dist) in 8 patients with MM (mean age 49 years and mean BMI 24 kg/m2 ) and 8 healthy controls (mean age 45 years and mean BMI 25 kg/m2 ) and the slice where most had the largest CSA was used as the ROI for further analysis. 2.4.3

Analysis of calf

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In the calf, the largest CSA of both the anterior and posterior muscle groups are at the proximal 33 % of the length of tibia (18). The length of tibia was measured using the localizer from the top of

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the right medial condyle of tibia to the base of the medial malleolus. As for the analysis of thigh

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muscles, we used Dixon sequences to manually outline the anterior compartment (tibialis anterior,

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extensor digitorum longus and extensor hallucis longus), the deep posterior compartment (tibialis posterior, flexor digitorum longus and flexor hallucis longus), peroneus longus et brevis, the medial

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gastrocnemius and the compartment including the lateral gastrocnemius and soleus. All data from plantar flexors were merged afterwards to minimise the amount of intermuscular fat. Proximal-distal gradient in fat content

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2.4.4

In some muscle diseases, the fat content may vary with the length of the muscles, making the CSA

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from a single slice less descriptive of the level of muscle affection. To investigate this, the fat content in the anterior and posterior group of the thigh was measured in 7 patients with MM at 35

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%, 40 %, 50 %, 60 % and 65 % of the length of femur. The fat content in the anterior and posterior group of the calf was measured in 33 patients with MM at 18 %, 33 % and 45 % of the length of tibia.

2.5 S TATISTICS MM patients were analysed by group and by subgroups.

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Repeated measure analysis of variance (repeated measure ANOVA) was used for comparing fat content through the muscles of the calf and thigh in patients with MM.

Welch sample t-test was used for comparing fat content, PT, CSA, CCSA, PT/CSA and PT/CCSA

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between patients with MM and controls. It was further used for comparing PT/CCSA between patients with high and low mutation load. When data were not normally distributed or had unequal

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variance, logarithmic transformation was performed if possible; otherwise a Kruskall Wallis test

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was used. P-values were Bonferroni corrected for PT/CSA and PT/CCSA. ANOVA was used for comparing the above measures between the subgroups of MM and controls.

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P-values were Bonferroni corrected.

ANOVA was used for investigating the relation between fat fractions and creatine kinase (CK)

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versus contractility. Logarithmic transformation was used when data was not normally distributed.

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Analysis of covariance (ANCOVA) was used for comparing linear regression lines between MM

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and controls. P-values were Bonferroni corrected.

P-values < 0.05 were considered significant.

3 RESULTS 3.1 SUBJECTS

One CPEO patient was excluded from analysis due to involuntary muscle contractions during scanning, which disturbed the MRI images. Another patient with CPEO was excluded due to

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diabetes with complications as neuropathy that might disturb the results. These patients are not presented in this paper. Two CPEO patients were excluded from the thigh analysis, because of a software malfunction that mixed Dixon fat and water images. Two patients with private mutations were too weak to use the muscle dynamometer at the ankle level, and could therefore not contribute

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with data for calf analyses. One patient with right-sided paresis after polio as a child had no affection of the left side, why this side was tested. One patient was excluded from the analysis of

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the importance of mutation load, since she carried a homoplasmic variant (19).

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All patients had mitochondrial myopathy without any symptoms from PNS or CNS that could disturb the results. One patient with encephalopathy (subject # 35) had exclusive cognitive

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symptoms, one patient with multiple sclerosis (MS, subject # 22) had been in a stable phase for

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many years without MS treatment and had never had a motor attack, one patient with systemic lupus erythematosus (subject # 11) had only had skin affection and was asymptomatic at the time of

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study, one patient with diabetes (subject # 15) was newly examined for diabetic complications without finding neuropathy or any other complications and another patient with diabetes (subject #

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10) had no clinical symptoms.

28 patients out of 35 had a muscle biopsy taken at the time of diagnosis. Neurogenic involvement

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was evaluated by looking for groups of angular, atrophic fibers and type grouping, which were not found in muscle biopsies of the patients. EMG and NCV were performed previously in 10 patients, all of which had either normal or myopathic EMG and normal NCV. None showed neuropathic affection.

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3.2 REGION OF INTEREST AT THIGH LEVEL The largest CSA of the posterior group was found at 65 % of the length of femur (prox-dist) and the anterior group at 40 % (Figure 1), which agrees

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with former findings (16,20).

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3.3 PROXIMAL-DISTAL GRADIENT IN FAT

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CONTENT

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There was no difference in fat content at the three levels of plantar flexors and five levels of thigh

Figure 1: MRI locations of cross-sectional areas of lower limb muscles.

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muscles (Figure 2). There was a small significant difference in dorsal flexors, but it is not regarded

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as being of any clinical importance.

Three slice levels were chosen at thigh level to capture the largest diameter of the three muscle groups assessed. Slice 1: CSA of the anterior thigh group (40 % of the length of femur, prox-dist). Slice 2: CSA of the posterior thigh group (65 %). Slice 3: anterior and posterior calf muscle groups (33% of the length of tibia, prox-dist).

3.4.1

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3.4 MUSCLE CONTRACTILITY

Characteristics of muscle groups

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As compared to healthy controls, patients with MM had a significantly higher muscle fat fraction and lower PT, CSA and CCSA (Figure 3 A-D).

Dividing MM into subgroups of CPEO and m.3243A>G point mutations did not change the conclusions (Figure 3

A-D).

Patients with

Figure 2: Fat distribution in thigh and calf. Fat fractions were compared at five levels in the thigh and at three levels in the calf. Level 1: 35 % and 18 % of the length of femur and tibia, respectively (prox-dist). Level 2: 40 % and 33 % of the length of femur and tibia, respectively. Level 3: 50 % and 45 % of the length of femur and tibia, respectively. Level 4: 60 % of the length of femur. Level 5: 65 % of the length of femur. Arrows are SD.

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m.3243A>G tended to be less affected than patients with CPEO. 3.4.2

PT/CSA and PT/CCSA

The specific force, calculated as the maximal PT divided by the CSA, was significantly reduced in all muscle groups in patients with MM as compared to healthy controls (figure 3E). PT/CCSA was

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also significantly reduced in patients with MM vs. healthy controls in all muscle groups, except the knee extensors (Figure 3F).

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PT/CSA and PT/CCSA were reduced in patients with CPEO in calf muscles, whereas patients with

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m.3243A>G only had reduced PT/CSA and PT/CCSA in plantar flexors, and showed normal

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contractility in the other muscle groups tested. There was found no significant difference in

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PT/CCSA between patients with CPEO and m.3243A>G.

There was no relationship between mutation load and PT/CCSA when comparing patients with low

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mutation load in skeletal muscle (CPEO with mutatio n load < 50 % and all other patients with

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mutation load < 70 %) and patients with high mutation load (Figure 3G).

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Figure 3: Muscle characteristics. A) pea k torque, B) fat fractions, C) CSA, D) CCSA, E) PT/CSA a nd F) PT/CCSA a re shown for healthy controls, patients with MM a nd the subgroups consisting of patients with CPEO a nd the mutation m.3243A>G. The relation between PT a nd CCSA for pa ti ents with MM wi th high a nd low mutation load (G)). Si gnificantly di fferent from control * i ndicates p<0.05. Arrows indicate s tandard errors. Do: dorsal, ext: extensors, flex: flexors, Pl: plantar.

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3.4.3

Linear regression models

A linear relation between PT and CCSA was found in patients with MM and healthy controls for knee flexors, knee extensors and dorsal flexors (Figure 4). This relation was not found in plantar flexors for patients with MM.

showed equal slopes in all muscle groups that had a linear relation. Impact of fat fractions and CK on contractility

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3.4.4

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There was no significant relation between CK and

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contractility. A significant relation between fat fraction and

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Linear regression models comparing PT and CCSA in patients with MM and healthy controls

contractility was found in knee flexors and plantar and

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dorsal flexors (Figure 5). This relation was not found in

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knee extensors.

Figure 4: Linear regression models of peak torque and contractile cross-sectional areas. Li near regression between PT a nd CCSA for knee fl exors

Figure 5: Impact of fat fractions on contractility. The rel ation between PT/CCSA a nd fat fractions i n knee fl exors, -extensors, plantar flexors and dorsal flexors i n pa ti ents with MM.

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4 DISCUSSION 4.1 MUSCLE CONTRACTILITY It is a well-known phenomenon that patients with mitochondrial myopathies fatigue during

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sustained muscle work, due to shortage of energy delivery for contraction. However, we showed

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that patients with MM had lower peak torque and smaller CSAs than healthy controls. This is perhaps not surprising considering that we also found a higher fat fraction in patients with MM, but

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importantly, when we corrected for fat content in muscle using Dixon MRI-technique, contractility

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of most leg muscles was still significantly perturbed. This points to a fixed prob lem with muscle contractility.

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Although this finding was universally found in all subgroups, there were also some differences among groups. Thus, contractility was only reduced in calf muscles of patients with CPEO and in

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plantar flexors of patients with the m.3243A>G mutation. This was not due to differences in severity of muscle affection among the groups, since the comparison of patients with high and low

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mutation load in skeletal muscle showed no difference in contractility. We do not believe that this result is affected by not knowing the mutation load in all the patients, since the patients with known

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and unknown mutation load showed similar characteristics regarding age, BMI and contractility in the four muscle groups.

We showed that the contractility decreases with the amount of intramuscular fat in all muscle groups having affected contractility, thereby explaining some of the decreased contractility in patients with MM. It further might explain some of the difference in contractility among groups, since patients with the m.3243A>G mutation had normal amounts of fat and normal contractility in their dorsal flexors, whereas patients with CPEO had high amounts of fat a nd reduced contractility in this muscle group.

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The cause of the different pattern of contractility among genotypes and muscle types may relate to variability in the distribution of muscle fibre types. Type I fibres have more mitochondria than type II fibres, which gives them higher levels of the intramitochondrial protein cyclophilin D (CypD). In MM, CypD is elevated (12). It regulates the mitochondrial permeability pore, that brings Ca2+ to its

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equilibrium. During a contraction Ca2+ increases in the cytoplasm thereby opening the permeability pore that brings Ca2+ inside the mitochondria (12). It is believed that this reduces the contractility.

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Former studies have shown that calf muscles contain more type I fibres than thigh muscles, and

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particularly knee extensors have the lowest fractions of type I fibres (20). Thus, a plausible explanation for worse muscle contractility in calf vs. thigh muscles could be this difference in fibre

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type distribution.

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The contractility could further have been affected by mitochondrial neuropathy. Mitochondrial neuropathy impairs the innervation of muscles, thereby reducing contractility. It more frequently

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affects the posterior tibial nerve (22), which could explain why the contractility was especially affected in the plantar flexors. However, the patients in this study did not show any clinical signs of

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neuropathy (for example numbness or burning sensations), and former muscle biopsies and nerve conduction velocity tests from a subgroup were without evidence of a neurogenic affection, making

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this theory unlikely.

In accordance with the severely affected contractility in calf muscle, there was no linear relation in plantar flexors between PT and CCSA in patients with MM. This relation was found in thigh muscles and dorsal flexors and suggests that the plantar flexors are more affected than other leg muscles.

4.2 PROXIMAL-DISTAL GRADIENT IN FAT CONTENT When estimating the muscle size using the CSA calculated from MRI, one needs to consider from which and how many slices it should be estimated. If there is a gradient in fat content, using the

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CSA from a single slice will give a bad measurement of the muscle size. In this case, either many slices or a calculation of the muscle volume will be better indicators. These methods, however, are more difficult and time consuming than using a single slice. The proximal-distal gradient in fat content of muscles in patients with MM has not yet been investigated. In this study, we show that

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there is no or minimal gradient in fat content in knee flexors, knee extensors, ankle dorsal flexors and ankle plantar flexors, and that the CSA from a single slice therefore can be used as an indicator

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of muscle size in patients with MM.

In conclusion, we show that patients with MM not only have fatigue and failing muscle

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contractility during prolonged muscle work, but also suffer from impaired fixed muscle contractility

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that partly relates to the distribution of intramuscular fat and partly relates to still unknown intrinsic changes in the musculature of patients with MM.

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In future research, it could be interesting to investigate the correlation between type 1 and type 2 fibres and contractility and whether there is a correlation between the number of ragged red fibres

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and contractility.

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5 ACKNOWLEDGEMENTS We wish to thank Poul-Henrik Mejer Frandsen, Department of Diagnostic Radiology, Rigshospitalet, for his assistance setting up the MRI protocol.

6 FUNDING The work was funded by Aase and Ejnar Danielsens Foundation, reference no: 10-001699. There are no competing interests to declare.

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