Just Accepted by International Journal of Neuroscience

Dysferlinopathy: Mitochondrial Abnormalities in Human Skeletal Muscle Fuchen Liu, Jianwei Lou, Dandan Zhao, Wei Li, Yuying Zhao, Xiulian Sun, Chuanzhu Yan doi:10.3109/00207454.2015.1034801

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Abstract Purpose Mitochondrial defects have been associated with a series of muscular diseases. Dysferlinopathy, however, has been rarely reported with mitochondrial dysfunction. Here we report a cohort of dysferlinopathy patients with mitochondrial abnormalities found in muscle. Methods Clinical data and muscle pathologies of 9 cases with dysferlinopathy were retrospectively studied. mtDNA copy number, protein levels and activities of mitochondrial enzyme complexes were assayed. Results 9 patients were diagnosed as having dysferlinopathy by DYSF sequencing and quantification of dysferlin levels in muscle homogenates. Muscle biopsies exhibited dystrophic changes (n D 9), ragged red fibers (n D 9) and cytochrome c oxidase deficient fibers (n D 9). mtDNA copy number increased significantly in 56% (15/27) of fibers with mitochondrial histology. Protein levels of complex IV subunits II (n D 5), complex III subunit core 2 (n D 2) and complex I NDUFB1 (n D 1) decreased. Impaired activities of complexes I, III and IV were observed in 56%, 33% and 78% of subjects and the activities were reduced by 21%, 18% and 40%, respectively. Besides, loss activities of complexes I/IV and decreased ATP level were also found in fibroblasts from dysferlinopathy. Conclusion Prominent mitochondrial abnormalities are common pathological findings in muscle from dysferlinopathy. Our data indicated that mitochondria may play a significant role in the progression of dysferlinopathy and also highlighted the potential of mitochondrial protective drugs in rescuing the symptoms of dysferlinopathy.

© 2015 Informa Healthcare USA, Inc. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.

Publisher: Taylor & Francis Journal: International Journal of Neuroscience

1

EP

Fuchen Liu1,2 , Jianwei Lou1, Dandan Zhao1, Wei Li1, Yuying Zhao1, Xiulian Sun3, Chuanzhu Yan1,4,5

Department of Neurology, Qilu Hospital of Shandong University; 2Department of Neurobiology and Kavli

C

Institute for Neuroscience, Yale University School of Medicine; 3 Otolaryngology Lab, Qilu Hospital of Shandong University; 4 Key Laboratory for Experimental Teratology of the Ministry of Education, School of Medicine,

AC

Shandong University; 5Brain Science Research Institute, Shandong University

Correspondence: Dr C Yan, Department of Neurology, Qilu Hospital of Shandong University, Key Laboratory for Experimental Teratology of the Ministry of Education, Brain Science Research Institute, Shandong University,

ST

China. No. 107, West Wenhua Road, Jinan, 250012, China. E-mail: [email protected]

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Dysferlinopathy: Mitochondrial Abnormalities in Human Skeletal Muscle

TE D

DOI: http://dx.doi.org/10.3109/00207454.2015.1034801

1

Abstract

Purpose Mitochondrial defects have been associated with a series of muscular diseases. Dysferlinopathy, however, has been rarely reported with mitochondrial dysfunction. Here we report a cohort of dysferlinopathy patients with

TE D

mitochondrial abnormalities found in muscle.

Methods Clinical data and muscle pathologies of 9 cases with dysferlinopathy were retrospectively studied.

Results 9 patients were diagnosed as having dysferlinopathy by DYSF sequencing and quantification of dysferlin

EP

levels in muscle homogenates. Muscle biopsies exhibited dystrophic changes (n=9), ragged red fibers (n=9) and

cytochrome c oxidase deficient fibers (n=9). mtDNA copy number increased significantly in 56% (15/27) of fibers with mitochondrial histology. Protein levels of complex IV subunits II (n=5), complex III subunit core 2 (n=2) and

C

complex I NDUFB1 (n=1) decreased. Impaired activities of complexes I, III and IV were observed in 56%, 33% and 78% of subjects and the activities were reduced by 21%, 18% and 40%, respectively. Besides, loss activities of

AC

complexes I/IV and decreased ATP level were also found in fibroblasts from dysferlinopathy.

Conclusion Prominent mitochondrial abnormalities are common pathological findings in muscle from dysferlinopathy. Our data indicated that mitochondria may play a significant role in the progression of dysferlinopathy and also highlighted the potential of mitochondrial protective drugs in rescuing the symptoms of

ST

dysferlinopathy.

Key Words: Muscle biopsy; Mitochondrion; Ragged red fibers; CCO-deficient fibers; Dysferlinopathy; Muscular dystrophy

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

mtDNA copy number, protein levels and activities of mitochondrial enzyme complexes were assayed.

2

Introduction As the main cellular energy generators, mitochondria produce ATP through oxidative phosphorylation or electron transport chain. The copy number of mitochondrial DNA (mtDNA) can vary depending on the energy needs of a cell[1] and oxidative stress conditions[2]. Either nuclear or mitochondrial genome mutations can lead to

TE D

dysfunction of the mitochondrial respiratory chain and result in the damage of energy generation. Moreover, mitochondrial defects have been associated with a series of muscular diseases, such as Duchenne muscular

dystrophy (DMD)[3, 4] and Ullrich congenital muscular dystrophy (UCMD)[5]. Dystrophic muscle fibers lead to

sarcoplasmic reticulum (SR). As mitochondria are closely associated to the sites of calcium entry into the cell and

EP

of internal Ca2+ release required for mitochondrial Ca2+ uptake[6], excessive Ca2+ in cytoplasm will thus activate

mitochondrial Ca2+ transport mechanisms, resulting in large Ca2+ loading in mitochondria and subsequently,

C

mitochondrial swelling, loss of mitochondrial membrane integrity and mitochondrial dysfunction[4].

Dysferlin, encoded by the DYSF gene, is implicated in vesicle fusion, trafficking and membrane repair[7]. Using

AC

laser wounding method, researchers found dysferlin-mediated membrane fusion was triggered by Ca2+ influx[8]. Moreover, recent advances in the study of dysferlin-deficient mouse models have highlighted dysferlin’s association with Ca2+ signaling proteins in T-tubules[9]. Researchers found that dysferlin was preferentially localized within the T-tubule, and could stabilize stress-induced Ca2+ signaling. In addition to T-tubule, several

ST

studies also demonstrated that dysferlin was localized to Golgi, endoplasmic reticulum (ER) and SR, contributing to the maintenance of Ca2+ homeostasis in muscle fibers[10-12]. Mutations in DYSF gene cause three different phenotypes of muscular dystrophies: limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM) and distal myopathy with anterior tibialis, commonly referred to as dysferlinopathy[13, 14]. Muscle biopsy

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

increased Ca2+ permeability into muscle cell and consequently dysregulate Ca2+ signaling at T-tubule and

of dysferlinopathy shows non-specific myopathic features, such as abundant necrotic fibers, regenerating fibers, inflammatory infiltrates and later connective tissue increase. Because of the established relationship between dysferlin and calcium homeostasis in T-tubules and SR, skeletal muscle from dysferlinopathy patients may display altered calcium dynamics. As dysregulated Ca2+ signaling at T-tubule and SR can lead to mitochondrial damage in

DMD[3, 4] and UCMD[5], it is reasonable to speculate that the susceptibly damaged T-tubules and SR in dysferlinopathy may also cause mitochondrial dysfunction. In addition, one recent study identified the alternative first exon of dysferlin encoding a putative mitochondrial targeting signal[15]. More interestingly, the other study found that dysferlin was localized in mitochondria[10]. The direct association between dysferlin and mitochondria 3

further promotes us to discuss whether patients with dysferlinopathy would exhibit abnormally mitochondrial metabolism.

In 2011, Gayathri et al[16] reported ragged red fibers (RRFs) in skeletal muscle of dysferlinopathy. However,

TE D

systematic study aimed at investigating biochemical changes in mitochondrial function in dysferlinopathy is still

sparse. In the current study, we reported 9 cases with dysferlinopathy displayed a higher proportion of RRFs and cytochrome c oxidase (CCO) deficient fibers in muscle pathologies. Upon detailed biochemical investigations, we

EP

Materials and Methods

Patient recruitment 9 patients with dysferlinopathy were studied. Patients were diagnosed according to clinical characteristics, muscle biopsy, dysferlin protein analysis in muscle homogenates and DYSF gene sequencing. 4

C

patients with Duchenne muscular dystrophy (DMD) and 4 patients with Becher muscular dystrophy (BMD), diagnosed according to clinical characteristics, muscle biopsy and dystrophin immunostaining in muscle specimens,

AC

were recruited as dystrophic (diseased) controls. Muscle samples from 4 unaffected subjects in whom the diagnosis of a muscle disease was excluded by clinical, histologic, and EMG criteria served as normal controls.

Muscle biopsy At the time of diagnosis, an open muscle biopsy was obtained. Muscle specimens were frozen in

ST

isopentane, cooled in liquid nitrogen and stored at -80℃. For histological examinations, 8µm frozen sections were stained with hematoxylin and eosin (H&E), NADH-tetrazolium reductase (NADH-TR), periodic acid Schiff (PAS) and oil red O (ORO). Biopsied samples were also screened for mitochondrial pathologies using modified Gomori trichrome stain (mGT), COX/SDH double stain, and succinate dehydrogenase (SDH) stain.

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

first demonstrated the potential mitochondrial dysfunction in dysferlinopathy.

Immunostaining Anti-dysferlin (Novocastra, HAMLET-CE) and dystrophin (Novocastra, DYS1, DYS2 and DYS3) immunostaining were performed in muscle specimens. Anti-mitochondria (Abcam, ab3298) immunostaining was carried out in muscle samples exhibiting RRFs. For mtDNA detection, anti-DNA (Milipore, MAB3299) immunofluorescent staining was performed. Fluorophores were 488 goat anti-rabbit IgG antibody (Life Technologies, A-11034).

Mutation analysis DNA was extracted from muscle specimens and sequence analysis of DYSF gene after PCR 4

amplifications were performed using previously described primers[17]. The identified mutations were further evaluated in 100 controls to rule out polymorphisms.

Western blot Protein level of dysferlin in muscle homogenates was tested by western blot. GAPDH level was used

TE D

as internal reference. Relative contents of mitochondrial respiratory chain complexes were assessed using total OXPHOS human wb antibody cocktail (Abcam, ab110411) and anti-NDUFB1 antibody (Proteintech Group,

16902-1-AP). Equal loading was confirmed with the porin (Abcam, ab15895) level. Detection and quantification

EP

Laser capture microdissection and DNA isolation 10μm thick RRFs or CCO-deficient fibers were isolated using the LEICA LMD 7000 laser capture microdissection system. Before microdissection, the tissue sections were stained with CCO/SDH and subjected to an ethanol and xylene dehydration. Total DNA was extracted from single

C

fiber section according to the protocol described by Khrapko, et al.[18] and Entela Bua, et al.[19].

AC

Real-time PCR The mtDNA copy number was determined by Real-time PCR. The forward primer 5’-TAACCCAAGTCAATAGAAGCC-3’ and reverse primer 5’-CTAGAGGGATATGAAGCACC-3’ of 12S rDNA encoded by mtDNA were used to amplify the mtDNA. The forward primer 5’-TGCACCACCAACTGCTTAGC-3’ and reverse primer 5’-GGCATGGACTGTGGTCATGAG-3’ of GAPDH were used to amplify a 87-bp nuclear

ST

DNA product. Quantification of relative copy number differences was carried out using both analysis of the difference in threshold amplification between mtDNA and nuclear DNA (delta delta C(t) method), and analysis with a standard curve of a reference template.

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

were performed with the Li-Cor Odyssey imaging system.

Biochemical studies Biochemical studies were performed to determine the respiratory chain enzyme activities. Mitochondria were isolated from fibroblasts using mitochondria isolation kit (Solarbio, China). For mitochondrial lysis, the tissue mitochondria lysis kit (GENMED, China) was used. Activities of complex I, III and IV were measured using the animal mitochondrial respiratory chain complexes I, III and IV activities quantitative determination kit (GENMED, China). These activities were expressed as a percentage of citrate synthase activity.

Results Clinical data and muscle pathologies Table 1 lists detailed clinical features of patients and controls. There were 5 5

males and 4 females in dysferlinopathy group. The mean age of onset was 25±9.3 years, ranging from 12 to 42 years, and mean course was 7.6±5.8 years, ranging from 2-20 years. 6 suffered from LGMD2B and other 3 presented a MM phenotype. Genetic studies of DYSF showed these 9 cases harbored mutations including 1 with homozygous mutation, 3 with compound heterozygous mutations and 5 with single heterozygous mutations (Table

TE D

2). 12 kinds of mutations, including 8 novel mutations which were absent in 100 healthy individuals, were found.

Western blot analysis displayed no expression of dysferlin in patients’ muscles, although in 5 patients (Patient

Pathological features are summarized in Table 3. Muscle specimens from dysferlinopathy showed dystrophic

EP

changes, such as variation in fiber diameters, necrosis with extensive phagocytosis and regenerating fibers (Figure 1B-a). Perivascular inflammatory infiltrates were seen in 2 patients (Patient #Dysf8 and #Dysf9) (Figure 1B-a). Fibers with deposits of basophilic granular material in the subsarcolemma (Figure 1B-b), accounting for 1%～5%,

C

were observed in all patients when H&E staining was performed. NADH-TR staining only revealed nonspecific changes. ORO staining exhibited mild lipid droplets in 2 patients (Figure 1B-f). PAS staining was unremarkable.

AC

Immunostaining of dysferlin showed no dysferlin expression in these subjects.

Muscle pathologies of DMD and BMD exhibited variation in fiber size, obvious necrotic and regenerating fibers (ranging from 9% to 22%) and proliferation of interstitial connective tissue. Other staining only revealed

ST

non-specific findings. Immunostaining of dystrophin showed dystrophin deficiency in these subjects.

Mitochondrial histopathology In addition to the dystrophic changes, typical RRFs, ranging from 1% to 5%, were observed on MGT staining in all dysferlinopathy subjects (Figure 1B-c). These RRFs were stained blue (called

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

#Dysf5, #Dysf6, #Dysf7, #Dysf8 #Dysf9) only single heterozygous mutation was identified (Figure 1A).

ragged blue fibers, RBFs, accounting for 1%-8%) when employing SDH staining (Figure 1B-d). CCO-deficient fibers (ranged from 1% to 10%), showing reduced or no CCO signal, were evident on combined CCO/SDH staining (Figure 1B-e). No positive correlations between the percentage of RRFs or CCO-deficient fibers and the age of onset, the duration of illness or the severity of muscle pathologies were found. Immunofluorescent staining of mtDNA displayed particulate pattern in several CCO-deficient fibers. Particulate immunostaining of mtDNA in RRFs was more intense, indicating mitochondria duplication (Figure 1B-g~i). No RRFs or CCO-deficient fibers were observed in DMD, BMD.

6

mtDNA copy number Quantifications of mtDNA copy number were performed in several single muscle fibers from dysferlinopathy (n=9), DMD (n=2), BMD (n=2) patients and control subjects (n=2). 1 RRFs, 2 CCO-deficient fibers and 1 fiber with normal histology were microdissected from each dysferlinopathy subject. Thus, 36 fibers (9 RRFs, 18 CCO-deficient fibers and 9 fibers with normal histology) were collected from these 9 patients. As

TE D

diseased and normal controls, fibers exhibited normal histology were also dissected from DMD, BMD and normal

individuals. A significant increase of the mtDNA copy number in 15 single fibers (including 6 RRFs and 9 CCO-deficient fibers) was found in patients with dysferlinopathy, which ranged from 163% to 736% (Table 4).

No mtDNA depletion was detected. We failed to find a significant change of mtDNA copy number in most fibers

EP

from DMD and BMD.

Quantification of mitochondrial enzyme complexes In dysferlinopathy group (Figure 2A and 2C), protein levels

C

of mitochondrial complex II (FeS subunit) and V (ATP synthase α-subunit) were in normal range compared to normal controls. Contents of complex I (subunit NDUFB1) were normal in almost all the patients except in patient

AC

#Dysf2. However, lower levels of complex IV (subunit II) and complex III (subunit core 2) were detected in 5 (Patient #Dysf1, #Dysf2, #Dysf4, #Dysf7 and #Dysf9) and 2 (Patient #Dysf2 and #Dysf8) subjects respectively. As diseased controls, protein levels of mitochondrial enzyme complexes were also detected in muscle homogenates from patients with DMD and BMD. Although only a few patients exhibited decreased levels of complex I (subunit

ST

NDUFB1) and complex II (FeS subunit), lower expression of complex III (Core 2 subunit), IV (FeS subunit) and V (ATP synthase α-subunit) were displayed in almost all DMD and BMD cases (Figure 2B and 2C).

Mitochondrial enzyme activities Mitochondrial enzyme complexes activities in fibroblast from patient #Dysf2

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

mtDNA copy numbers in other 3 RRFs, 9 CCO-deficient fibers and 9 fibers with normal histology were normal.

were detected. Compared to normal control, activities of mitochondrial enzyme complex I (0.210±0.008) and IV

(0.836±0.026) were reduced to 72% and 63% of the control value (control complex I: 0.292±0.039; complex IV: 1.327±0.045) respectively (Figure 3A). There was no statistically significant difference of complex III between patient (1.005±0.011) and control (1.071±0.068). To determine whether the observed mitochondrial dysfunction had an effect on cellular bioenergetics, intracellular ATP level was measured. Results showed the ATP level was reduced to 72% of the control value (44.916 nmol/mg protein in patient vs 62.384 nmol/mg protein in control) (Figure 3B).

7

Then, mitochondrial enzyme complexes activities in muscle homogenates were examined. Results are presented in Table 5 and Figure 3C. Compared to normal controls, the activity of complex I in skeletal muscle of dysferlinopathy was significantly reduced by 21% (0.201±0.066 vs 0.254±0.016). Complex IV activity was also significantly decreased by 40% (0.664±0.196 vs 1.100±0.173). However, the activity of complex III was mildly

TE D

reduced only by about 18% (1.101±0.356 vs 1.344±0.084), when compared with the normal control subjects. Of these 9 subjects, 2 only showed reduced activity of complex IV. 1 patient only presented impaired activity of

complex III. Reduced activities of complex I and IV were observed in 3 subjects, and 2 showed underactivity of

samples from DMD and BMD. Consistent with previous reports[20] and the results of quantification of

EP

mitochondrial enzyme complexes, mitochondrial dysfunction was also evident in DMD and BMD, as indicated by loss of enzyme histochemistry signals of the mitochondrial activities I, II and III in some cases.

C

Discussion

The diagnosis of dysferlinopathy has relied primarily on clinical phenotype, high serum creatine kinase levels, and

AC

immunostaining for dysferlin on muscle biopsies. DNA analysis is the ultimate way to confirm the diagnosis of dysferlinopathy. In practice, it remains challenging to give the diagnosis by DNA analysis. It is not uncommon to only find one of the two expected mutations of the DYSF gene. For those patients only one mutation was identified, Gal et al.[21] highlighted the importance of the protein analysis in the diagnosis of dysferlinopathy. Actually,

ST

western blot of dysferlin has been put forth as a simple and effective screening tool for dysferlinopathy[22, 23]. Thus, according to dysferlin protein quantification and gene sequencing results, all these 9 patients in our study received the diagnosis of dysferlinopathy.

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

complexes I, III and IV. As diseased controls, respiratory chain enzyme activities were also studied in muscle

To our knowledge, only one study reported RRFs in dysferlinopathy[16]. However, no biochemical studies of mitochondrial enzyme activities were performed in their study. Thus, our research is the first study demonstrating biochemically mitochondrial abnormalities in dysferlinopathy. The findings of mitochondrial damage in muscle suggest a mitochondrial dysferlinopathy. Upon biochemical evaluations of mitochondrial enzyme activities in dysferlinopathy, DMD and BMD, we found mitochondrial abnormalities were not unique to dysferlinopathy; on the contrary, mitochondrial changes may be secondary to myofiber damage. In fact, the idea that mitochondrial dysfunction could be associated with dystrophic pathology was based on several studies in a variety of muscular dystrophies, such as in DMD[3, 4] and UCMD[5]. In DMD, deficiency of dystrophin muscle fibers could lead to 8

increased Ca2+ permeability into muscle fibers. Higher intracellular Ca2+ dysregulated calcium signaling at T-tubule and then promoted Ca2+ overload in mitochondria. In UCMD, deficiency of collagen IV affects cellular signaling pathway and impinges mitochondria and SR, causing increased transient openings of mitochondrial permeability transition pores and abnormal Ca2+ handling by the SR. Actually, the relationship between mitochondria and Ca2+

TE D

regulation in adult mammalian striated muscles was established many years ago. In muscle fibers, mitochondria are

in close proximity to the sites of calcium entry into the cell and of internal Ca2+ release, providing a prime real

estate for mitochondria to take up Ca2+ during normal cell activity or under several pathological conditions[6]. Thus,

mitochondrial dysfunction in muscle dystrophies. Dysferlin, comprised of multiple Ca2+ sensitive C2 domains, is

EP

implicated in vesicle fusion, trafficking, and membrane repair. Based on the observations that dysferlin accumulates

at wound sites in myofibers[24] and sea urchin embryos[25] in a Ca2+-dependent manner, it is thought that dysferlin mediates Ca2+-dependent membrane repair. Moreover, several recent studies demonstrated that dysferlin was

C

enriched in T-tubule membrane and the membrane of ER and SR, contributing to the maintenance of Ca2+ homeostasis in muscle[10-12]. Muscles from dysferlin-deficient mouse exhibited increased T-tubule damage

AC

and Ca2+ dysregulations[9]. As dysregulated Ca2+ signaling at T-tubule and SR are known to underscore mitochondrial damage in DMD and UCMD, it is reasonable to believe that Ca2+-dependent T-tubules and SR damage in dysferlinopathy may also cause mitochondrial dysfunction.

ST

Although impaired mitochondrial enzyme activities were observed in dysferlinopathy, DMD and BMD, only muscle biopsies from dysferlinopathy exhibited RRFs or CCO-deficient fibers. One possible explanation is that some more effective compensatory machineries, such as up-regulation of mitochondrial fission or mitochondrial mass, were initiated to offset the respiratory chain defect in dysferlinopathy. Significant increase of mtDNA copy

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

according to these previous studies, we concluded that Ca2+ dysregulation may play a prominent role in

number in several RRFs or CCO-deficient fibers indeed indicated a compensatory machinery might be initiated in dysferlinopathy. The other interpretation is that dysferlin itself may have certain relationship with mitochondria. A study conducted by Morree A et al.[15] found a large number of mitochondrial proteins localizing in the outer and inner mitochondrial membrane in the dysferlin protein complex and also identified the alternative first exon of dysferlin encoding a putative mitochondrial targeting signal. This study raised the possibility that dysferlin might be targeted to mitochondria. More interestingly, a recent study provided a direct evidence for dysferlin’s localization to mitochondria, in addition to the ER and Golgi networks where it predominates[10]. Thus, we inferred that mutant dysferlin protein in dysferlinopathy would disrupt the direct correlation between dysferlin and 9

mitochondria, eventually causing RRF or CCO-deficient fibers in dysferlinopathy.

Owing to mitochondrial vulnerability to several endogenous and exogenous factors, mitochondrial abnormalities in dysferlinopathy could also be secondary to other causes. Although aging can cause mitochondrial changes, it is

TE D

interesting to note that most of our cases are still young and the changes we observed are much more pronounced

than those described in healthy elderly individuals. Moreover, we couldn’t find any correlations between

mitochondrial abnormalities and the duration of the disease, the severity of muscle pathologies or inflammatory

EP

Generally, there is a positive correlation between mitochondrial enzyme activities and mtDNA copy number.

Tomomi et al.[26] reported decreased mtDNA copy number and lower enzymatic activity of complexes in myocardial infarction. The other study showed the depletion of mtDNA in muscle fibers was correlated with a

C

respiratory chain defect[27]. In our study, however, we found in dysferlinopathy, mtDNA copy number was increased significantly in several RRFs or CCO-deficient fibers. Similar to our results, a study also reported

AC

increased mtDNA copy number and decreased activities of electron transport chain complexes in the brain from subjects with autism[28]. We inferred that increased mtDNA copy number in dysferlinopathy might represent a mechanism to compensate for the lower activity of mitochondrial enzyme complexes. Moreover, it is known that an increase of mtDNA copy number can also be caused by oxidative stress[2]. Thus, the increase of mtDNA copy

ST

number in skeletal muscle from dysferlinopathy may be attributed to increased oxidative stress in this disease.

Conclusion

In this paper, we reported RRFs and CCO-deficient fibers in dysferlinopathy. The unquestionable evidence of

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

infiltrates.

mitochondrial abnormalities in dysferlinopathy indicate that mitochondria may play a significant role in the progression of the disease. Our study may open avenues for investigation of new therapeutics in this disease.

Declaration of interest: The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the paper

10

Reference 1.

Shay, J.W., D.J. Pierce, and H. Werbin, Mitochondrial DNA copy number is proportional to total cell DNA

2.

Liu, C.S., et al., Oxidative stress-related alteration of the copy number of mitochondrial DNA in human

under a variety of growth conditions. J Biol Chem, 1990. 265(25): p. 14802-7.

3.

TE D

leukocytes. Free Radic Res, 2003. 37(12): p. 1307-17.

Katsetos, C.D., S. Koutzaki, and J.J. Melvin, Mitochondrial dysfunction in neuromuscular disorders. Semin Pediatr Neurol, 2013. 20(3): p. 202-15.

4.

Deconinck, N. and B. Dan, Pathophysiology of duchenne muscular dystrophy: current hypotheses. Pediatr

5.

Bernardi, P. and P. Bonaldo, Dysfunction of mitochondria and sarcoplasmic reticulum in the pathogenesis of collagen VI muscular dystrophies. Ann N Y Acad Sci, 2008. 1147: p. 303-11.

Franzini-Armstrong, C., ER-mitochondria communication. How privileged? Physiology (Bethesda), 2007. 22: p.

EP

6.

261-8. 7.

Kerr, J.P., C.W. Ward, and R.J. Bloch, Dysferlin at transverse tubules regulates Ca(2+) homeostasis in skeletal

8.

McNeil, P.L. and T. Kirchhausen, An emergency response team for membrane repair. Nat Rev Mol Cell Biol,

muscle. Front Physiol, 2014. 5: p. 89. 2005. 6(6): p. 499-505.

Kerr, J.P., et al., Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane. Proc

C

9.

Natl Acad Sci U S A, 2013. 110(51): p. 20831-6.

Mueller, A.L., et al., Improved immunoblotting methods provide critical insights into phenotypic differences

AC

10.

between two murine dysferlinopathy models. Muscle Nerve, 2014. 50(2): p. 286-9. 11.

Flix, B., et al., Dysferlin interacts with calsequestrin-1, myomesin-2 and dynein in human skeletal muscle. Int J Biochem Cell Biol, 2013. 45(8): p. 1927-38.

12.

Fujita, E., et al., Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum Mol Genet, 2007. 16(6): p. 618-29.

Illa, I., et al., Distal anterior compartment myopathy: a dysferlin mutation causing a new muscular dystrophy

ST

13.

phenotype. Ann Neurol, 2001. 49(1): p. 130-4. 14.

Liu, J., et al., Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular

15.

de Morree, A., et al., Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal

dystrophy. Nat Genet, 1998. 20(1): p. 31-6. maintenance and integrity. PLoS One, 2010. 5(11): p. e13854.

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Neurol, 2007. 36(1): p. 1-7.

16.

Gayathri, N., et al., Dysferlinopathy: spectrum of pathological changes in skeletal muscle tissue. Indian J Pathol Microbiol, 2011. 54(2): p. 350-4.

17.

Aoki, M., et al., Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy.

18.

Khrapko, K., et al., Cell-by-cell scanning of whole mitochondrial genomes in aged human heart reveals a

Neurology, 2001. 57(2): p. 271-8.

significant fraction of myocytes with clonally expanded deletions. Nucleic Acids Res, 1999. 27(11): p. 2434-41. 19.

Bua, E., et al., Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am J Hum Genet, 2006. 79(3): p. 469-80.

20.

Ramadasan-Nair, R., et al., Mitochondrial alterations and oxidative stress in an acute transient mouse model of muscle degeneration: implications for muscular dystrophy and related muscle pathologies. J Biol Chem, 2014. 11

289(1): p. 485-509. 21.

Gal, A., et al., Challenges for the genetic screening in dysferlin deficiency--report of an instructive case and review of the literature. Clin Neuropathol, 2008. 27(5): p. 289-94.

22.

Ho, M., et al., A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi

23.

Ankala, A., et al., Diagnostic overview of blood-based dysferlin protein assay for dysferlinopathies. Muscle Nerve, 2014. 50(3): p. 333-9.

24.

Bansal, D., et al., Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature, 2003. 423(6936): p. 168-72.

25.

Covian-Nares, J.F., et al., Membrane wounding triggers ATP release and dysferlin-mediated intercellular

26.

Ide, T., et al., Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts

calcium signaling. J Cell Sci, 2010. 123(Pt 11): p. 1884-93. after myocardial infarction. Circ Res, 2001. 88(5): p. 529-35.

Moraes, C.T., et al., mtDNA depletion with variable tissue expression: a novel genetic abnormality in

EP

27.

mitochondrial diseases. Am J Hum Genet, 1991. 48(3): p. 492-501. 28.

Gu, F., et al., Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism. Transl Psychiatry, 2013.

ST

AC

C

3: p. e299.

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

TE D

myopathy. Ann Neurol, 2002. 51(1): p. 129-33.

12

Table 1 Summary of the clinical data of patients with dysferlinopathy and DMD/BMD

Sex

duration of

onset (years)

illness (years)

Proximal

Distal

Proximal

Distal

3

2

5

2

Upper limb

Lower limb

CK

Phenotype

3

1,433

LGMD2B

4

5,821

MM

4 5

2,478 3,250

LGMD2B LGMD2B

2

3,860

MM

5 4

4,900 2,343

LGMD2B LGMD2B

4

7,304

LGMD2B

4 2

3 4

5,615 12,220

MM DMD

4-

4

10,711

DMD DMD DMD

F

42

#Dysf 2

M

26

6

5

5

5

#Dysf 3 #Dysf 4

F M

17 34

12 2

5 5

5 5

3 2

#Dysf 5

M

12

20

4

4

3

#Dysf 6 #Dysf 7

F M

30 25

10 4

4 4

5 5

4 3

#Dysf 8

M

20

3

5

5

4

#Dysf 9

F

19

8

4

5

#DMD 1

M

7

3.5

4

3

#DMD 2

M

5

2

4

5

#DMD 3 #DMD 4

M M

3 4

0.5 2

#BMD 1

M

32

15

4

#BMD 2 #BMD 3

M M

35 29

18 10

4 4

#BMD 4

M

36

20

4

EP

#Dysf 1

15,270 20,174

4

4

4

8,787

BMD

4 3

3 3

4 4

9,103 5,011

BMD BMD

3

3

4

7,326

BMD

AC

C

No record No record

Muscle strength was assessed according to a modified Medical Research Council (MRC) muscle grading system.

ST

CK, serum creatine kinase ( normal value: 26-178IU/L)

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

No.

Muscle strength

Age of

TE D

Patients’

13

Table 2 Mutations identified in the DYSF gene Gene sequencing

#Dysf 1

Compound heterozygotes

cDNA change 1

Mutational event 1

c.2803G>A

missense

＊

#Dysf 2

Compound heterozygotes

c.610C>T

nonsense

#Dysf 3

Compound heterozygotes

c.5826C>A

nonsense

#Dysf 4

Homozygote

c.2268delG

Heterozygotes

frameshift

IVS13+2T>C

＊

splice

＊

splice

#Dysf 6

Heterozygotes

IVS13+2T>C

#Dysf 7

Heterozygotes

IVS26+1G>A

#Dysf 8

Heterozygotes

IVS12-2A>G

#Dysf 9

Heterozygotes

c.1656T>G

AC ST JU 14

c.3112C>T

＊

Mutational event 2 nonsense

c.2311C>T

nonsense

c.581C>A

missense

----

----

----

----

----

----

----

----

splice

----

----

nonsense

----

----

C

* mutation had been reported previously

splice

cDNA change 2

EP

#Dysf 5 Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

State

TE D

Patients’ No.

Table 3 Summary of the muscle pathological features of dysferlinopathy and DMD/BMD

3/5 3/4 2/2 5/8 1/1 3/5 2/2 1 1 -

lipid droplets

5 7 3 10 3 4 3 1 1 -

+, mildly +, mildly -

ST

Abbreviations: RRF, ragged red fiber; RBF, ragged blue fibers; CCO, cytochrome c oxidase

15

glycogen

TE D

+ + + + -

CCO deficient fibers （%）

EP

～7% ～10% ～6% ～10% ～8% ～11% ～5% ～10% ～7% ～30% ～25% ～28% ～17% ～22% ～15% ～9% ～12%

RRFs/RBFs （%）

C

Biceps brachii Gastrocnemius Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii Gastrocnemius Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii Biceps brachii

inflammatory infiltration

AC

#Dysf 1 #Dysf 2 #Dysf 3 #Dysf 4 #Dysf 5 #Dysf 6 #Dysf 7 #Dysf 8 #Dysf 9 #DMD 1 #DMD 2 #DMD 3 #DMD 4 #BMD 1 #BMD 2 #BMD 3 #BMD 4

Biopsied muscle

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Patients’ No.

necrotic or regenereating fibers (%)

-

#Dysf 4

#Dysf 5

ST

#Dysf 6

#Dysf 7

#Dysf 8

#Dysf 9

#DMD 1 #DMD 2 #BMD 1

mtDNA/nDNA 5703.667±100.202 829.523±113.080 7937.667±147.161 1101.219±47.189 4950.000±50.089 1135.899±56.374 913.333±110.151 1178.782±151.242 1126.544±65.023 4947.511±81.574 1765.021±162.558 972.333±37.786 4866.667±152.752 1118.667±74.768 1037.333±74.3931 1088.289±117.026 1177.087±59.908 4033.323±152.753 6307.289±189.255 1132.667±186.090 1121.681±38.566 1081.644±56.899 1331.277±24.151 1100.000±136.277 2915.414±177.021 959.523±221.443 4190.699±204.295 1092.270±114.233 3088.242±193.283 1958.210±71.882 3424.280±68.789 927.274±101.363 2556.201±85.983 7959.333±145.258 1079.028±35.796 1059.100±113.270 896.514±128.357 1113.570±142.240 988.419±104.276 1028.275±146.387 1013.258±151.077 1074.049±143.296

EP

C

#Dysf 3

Fiber pathology RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber RRF CCO-deficient fiber CCO-deficient fiber normal fiber normal fiber normal fiber 16 normal fiber normal fiber normal fiber normal fiber

AC

#Dysf 2

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Patients’ No. #Dysf 1

TE D

Table 4 Average mtDNA copy number from patients with dysferlinopathy, DMD/BMD and normal controls Note: Values in bold are significantly different (P＜0.05) from the respective values for controls

#BMD 2 #C1 #C2

normal fiber normal fiber normal fiber normal fiber normal fiber normal fiber

903.212±87.285 1121.277+102.388 1103.667±99.002 1028.15±70.314 1076.11±102.504 1174.333±137.129

TE D

EP C AC ST JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Note: Values in bold are significantly different (P＜0.05) from the respective values for controls

17

Table 5 Activities of mitochondrial respiratory chain complexes in skeletal muscle from patients with dysferlinopathy, DMD/BMD and normal controls Complex IV

Affected complexes

0.148±0.012 0.142±0.018 0.267±0.028 0.121±0.015 0.268±0.010 0.263±0.026 0.174±0.007 0.278±0.003 0.146±0.006 0.201±0.066 56%

1.453±0.068 0.646±0.045 1.289±0.034 0.745±0.109 1.348±0.057 0.521±0.039 1.315±0.022 1.308±0.040 1.287±0.033 1.101±0.356 33%

0.602±0.017 0.635±0.067 0.637±0.040 0.410±0.086 0.439±0.103 0.931±0.037 0.681±0.035 1.002±0.591 0.636±0.061 0.664±0.196 78%

I, IV I, III, IV IV I, III, IV IV III I, IV None I, IV

0.182±0.009 0.199±0.008 0.201±0.011

0.709±0.027 1.028±0.102 0.535±0.044 0.621±0.008 0.723±0.215 75%

0.555±0.013 0.607±0.088 0.378±0.102 0.699±0.005 0.560±0.135 100%

III, IV IV III, IV I, III, IV

IV III, IV I, III, IV I, IV

#DMD 1 #DMD 2 #DMD 3 #DMD 4 Mean±SD Affected subjects #BMD 1 #BMD 2 #BMD 3 #BMD 4 Mean±SD Affected subjects

0.220±0.026 0.241±0.005 0.145±0.011 0.101±0.012 0.177±0.065 50%

1.207±0.012 0.611±0.022 0.289±0.035 1.212±0.103 0.830±0.458 50%

0.659±0.103 0.599±0.009 0.715±0.019 0.695±0.033 0.658±0.048 100%

#C 1 #C 2 #C 3 #C 4 Mean±SD

0.263±0.011 0.231±0.105 0.258±0.015 0.265±0.100 0.254±0.016

1.363±0.588 1.267±0.069 1.291±0.075 1.453±0.088 1.344±0.084

1.018±0.022 1.255±0.026 1.231±0.028 0.896±0.035 1.100±0.173

Control

ST

EP

C

AC

0.131±0.013 0.179±0.033 25%

TE D

Dysferlinopathy

Complex III

DMD

Complex I

BMD

Patients’ No. #Dysf 1 #Dysf 2 #Dysf 3 #Dysf 4 #Dysf 5 #Dysf 6 #Dysf 7 #Dysf 8 #Dysf 9 Mean±SD. Affected subjects

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Note:

Values in bold are significantly different (P＜0.05) from the respective values for controls

18

TE D EP C AC ST

homogenates. #Dys1-9 are patients with dysferlinopathy; Subjects #C1-#C4 are normal controls B. Representative

JU

Int J Neurosci Downloaded from informahealthcare.com by Nyu Medical Center on 05/25/15 For personal use only.

Fig 1 Western blot and muscle pathologies in dysferlinopathy. A. Western blot of dysferlin from muscle

light microscopy images of muscle pathology. (a) Typical perivascular inflammatory infiltration (arrowhead) and necrotic fiber (arrow) on H&E staining. (b~d) Fibers with deposits of basophilic granular material along the sarcolemma (b, H&E staining), serial sections stained showed these fibers were typical RRFs (c, MGT staining) and RBFs (d, SDH staining). (e) CCO-deficient fibers on combined SDH/CCO staining. (f) Mild increase of lipid droplets in Dysf9 on ORO staining. (g) Immunostaining with anti-mitochondrial antibody. (h) Immunofluorescence staining with anti-DNA antibody, increased duplication of mitochondria in RRFs (arrowhead) and several CCO-deficient fibers (arrow) are shown. (i) Serial section stained with combined CCO/SDH.

19

TE D EP C AC ST

respiratory chain in muscle homogenates from dysferlinopathy (A) and DMD/BMD (B). C. Quantification of western blot results. Values represent mean±SD of 3 separate experiments, *p