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doi: 10.1152/japplphysiol.01096.2012114:161-171, 2013. First published 25 October 2012;J Appl Physiol

Martin Picard, Kathryn White and Douglass M. Turnbullthree-dimensional electron microscopy studyinteractions in skeletal muscle: a quantitative Mitochondrial morphology, topology, and membrane

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Mitochondrial morphology, topology, and membrane interactions in skeletalmuscle: a quantitative three-dimensional electron microscopy study

Martin Picard,1,2,3 Kathryn White,4 and Douglass M. Turnbull1,5,6

1Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UnitedKingdom; 2Department of Kinesiology and Physical Education, McGill University, Montreal, Canada; 3Center forMitochondrial and Epigenomic Medicine, The Children’s Hospital of Philadelphia and University of Pennsylvania,Philadelphia, Pennsylavania; 4EM Research Services, Newcastle University, Newcastle upon Tyne, United Kingdom;5Newcastle University Centre for Brain Ageing and Vitality, Institute for Ageing and Health, Newcastle University, Newcastleupon Tyne, United Kingdom; and 6Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health,Newcastle University, Newcastle upon Tyne, United Kingdom

Submitted 10 September 2012; accepted in final form 22 October 2012

Picard M, White K, Turnbull DM. Mitochondrial morphology, topol-ogy, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study. J Appl Physiol 114: 161–171, 2013.First published October 25, 2012; doi:10.1152/japplphysiol.01096.2012.—Dynamic remodeling of mitochondrial morphology through membranedynamics are linked to changes in mitochondrial and cellular function.Although mitochondrial membrane fusion/fission events are frequentin cell culture models, whether mitochondrial membranes dynami-cally interact in postmitotic muscle fibers in vivo remains unclear.Furthermore, a quantitative assessment of mitochondrial morphologyin intact muscle is lacking. Here, using electron microscopy (EM), weprovide evidence of interacting membranes from adjacent mitochon-dria in intact mouse skeletal muscle. Electron-dense mitochondrialcontact sites consistent with events of outer mitochondrial membranetethering are also described. These data suggest that mitochondrialmembranes interact in vivo among mitochondria, possibly to inducemorphology transitions, for kiss-and-run behavior, or other processesinvolving contact between mitochondrial membranes. Furthermore, acombination of freeze-fracture scanning EM and transmission EM inorthogonal planes was used to characterize and quantify mitochon-drial morphology. Two subpopulations of mitochondria were studied:subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibitedsignificant differences in morphological descriptors, including formfactor (means � SD for SS: 1.41 � 0.45 vs. IMF: 2.89 � 1.76, P �0.01) and aspect ratio (1.97 � 0.83 vs. 3.63 � 2.13, P � 0.01) andcircularity (0.75 � 0.16 vs. 0.45 � 0.22, P � 0.01) but not size(0.28 � 0.31 vs. 0.27 � 0.20 �m2). Frequency distributions formitochondrial size and morphological parameters were highlyskewed, suggesting the presence of mechanisms to influence mito-chondrial size and shape. In addition, physical continuities betweenSS and IMF mitochondria indicated mixing of both subpopulations.These data provide evidence that mitochondrial membranes interact invivo in mouse skeletal muscle and that factors may be involved inregulating skeletal muscle mitochondrial morphology.

skeletal muscle mitochondria; myofiber; outer mitochondrial mem-brane; scanning and transmission electron microscopy; mitochondrialreticulum

SKELETAL MUSCLE FIBERS PERFORM energetically demanding func-tions (contraction, rapid cycles of ion transport, gene expres-sion, and protein synthesis) requiring large amounts of ATP

supplied in large part by mitochondria.1 A unique and rela-tively recently discovered feature of mitochondria is theirability to fuse and divide, collectively known as mitochondrialdynamics (6, 39). Dynamic remodeling of mitochondria fromspheroid, bean-shaped structures to elongated tubules, and viceversa, is now understood as an integral aspect of mitochondrialbiology. Mitochondrial fusion and fission regulate the au-tophagy quality control system (35, 37) and apoptotic sensi-tivity (19, 38) and may act as a metabolic sensing mechanisminfluencing cell survival in response to nutritional stress (11,25, 30) and possibly muscle atrophy (31). Furthermore, thegenes encoding mitochondrial dynamics proteins have beenidentified and genetic disruptions of either mitochondrial fu-sion or fission components impair embryonic development (7,14), cause the accumulation of mitochondrial DNA impair-ments in muscle fibers (8), and lead to neuromuscular disordersin humans (40, 41). Other work also suggests that low levels ofthe mitochondrial fusion protein mitofusin 2 (Mfn2) impairskeletal muscle insulin sensitivity in diabetes (2, 12). Collec-tively, this evidence has been taken to imply that mitochondrianormally undergo fusion and fission in vivo and that disruptingthese processes, and therefore mitochondrial morphology, neg-atively impairs the function of skeletal muscle.

However, although mitochondria continuously undergomorphological changes in the order of seconds to minutes inthe cytosol of proliferating cultured cells (36), direct evidenceof mitochondrial membrane interactions (a prerequisite formitochondrial dynamics) in skeletal muscle fibers is scarce(24). Furthermore, methods to examine and quantify mitochon-drial shape in intact myofibers are required to determinewhether changes in mitochondrial morphology are linked tospecific physiological functions of skeletal muscle fibers.

Here, building from previous work by others (4, 16, 17, 22),we used a combination of freeze-fracture scanning electronmicroscopy (SEM) and resin-embedded transmission EM(TEM) in both longitudinal and transverse planes of mousesoleus muscle to characterize mitochondria topology and quan-tify their morphology. Our novel quantitative approach re-vealed that standard morphological parameters for both sub-sarcolemmal (SS) and intermyofibrillar (IMF) mitochondriadiffered extensively and were generally skewed in their distri-

Address for reprint requests and other correspondence: M. Picard, Center forMitochondrial and Epigenomic Medicine, The Children’s Hospital of Phila-delphia, Univ. of Pennsylvania, Colket Translational Research Bldg., Phila-delphia, Pennsylvania 19104 (e-mail: [email protected]).

1 This article is the topic of an Invited Editorial by David Hood and SobiaIqbal (12a).

J Appl Physiol 114: 161–171, 2013.First published October 25, 2012; doi:10.1152/japplphysiol.01096.2012.

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butions. In addition, several sites of mitochondrial membranesinteraction were apparent among and between IMF and SScompartments, contributing new evidence that mitochondrialmembranes dynamically interact in vivo, possibly to accom-plish fusion/fission (34), kiss-and-run (20), or for other phys-iological processes within intact muscle fibers.

METHODS

Animals and tissue collection. Eight-week-old female C57BL/6mice were studied. Mice were group housed with free access to foodand water until the morning preceding the experiments, when threemice derived from different litters were killed by cervical dislocationduring the dark phase of the light cycle (22:15). Within 1–1.5 minfollowing death, the soleus was dissected, cut into two equal halves,and fixed by immersion into fixative for either transmission electronmicroscopy (TEM) or scanning electron microscopy (SEM). Tissuesthereafter remained in fixative overnight at room temperature and�48 h at 4°C before processing for electron microscopy. All animalprocedures were approved by the University of Newcastle AnimalCare and Ethics Committee.

TEM. For TEM, half of the mouse soleus was immediately fixed ina 2% glutaraldehyde solution in 0.1 M cacodylate (TAAB LabEquipment) buffer, pH 7.4. Muscle samples were then postfixed in 1%osmium tetroxide (OsO4, Agar Scientific) for 1 h and dehydrated insequential steps of acetone (25%, 50%, 75%, and 100% twice) priorto impregnation in increasing concentrations (25%, 50%, 75%, and100% three times) of resin (TAAB Lab Equipment) in acetone over a24-h period. The half soleus was cut into smaller segments andembedded either in transverse (TS) or longitudinal (LS) orientation in100% resin at 60°C for 24 h. To verify orientation and section quality,1-�m-thick sections were cut and stained with 1% toluidine blue in1% borax. Ultrathin sections of 70 nm were subsequently cut using adiamond knife on a Leica EM UC7 ultramicrotome. Sections werestretched with chroloform to eliminate compression and mounted onPioloform filmed copper grids prior to staining with 2% aqueousuranyl acetate and lead citrate (Leica). Ultrathin sections were exam-ined on a Phillips CM 100 Compustage (FEI) transmission electronmicroscope, and digital micrographs were captured by an AMT CCDcamera (Deben).

To analyze SS mitochondrial morphology, muscle in the LS ori-entation was photographed at �19,000 magnification. Regions con-taining numerous SS mitochondria were selected for analysis. ForIMF mitochondria, muscle in the TS orientation was imaged at�7,900 magnification. To ensure optimal transverse orientation at thesubcellular level (relative to the Z-line) (see Fig. 1D), muscle fiberspresenting no more than two Z-lines separated by a minimum of10–15 �m were selected for analysis. For each animal, five to sixmitochondria-rich muscle fibers were analyzed in both TS and LS, forwhich at least two micrographs were captured, allowing the anal-ysis of �40 IMF and 40 SS mitochondria per myofiber. A total of505 SS and 653 IMF mitochondria were analyzed. The coefficientof variation (CV) for average mitochondrial size between myofi-bers was of 48.6% for SS and 15.6% for IMF mitochondria,whereas the CV between animals was 34.0% and 8.4%, respec-tively. For aspect ratio, a measure of mitochondrial shape, the CVbetween myofibers was of 12.4% for SS and 14.4% for IMFmitochondria, whereas the CV between animals was 2.5% and5.9%, respectively.

To quantify mitochondrial interactions among SS and IMF mito-chondria, micrographs of muscle photographed in the longitudinalorientation at �19,000 (SS) and �13,500 (IMF) were used. In theIMF compartment, a total of 750 Z-lines possessing mitochondria onboth sides, either as pairs of discrete organelles (Fig. 3A), as acontinuous mitochondrion (Fig. 3B), or as interacting organelles (Fig.3C), were analyzed, and the proportion of Z-lines spanned by acontinuous or interacting mitochondria is reported as a percentage of

the total. In addition, 437 SS and 667 IMF physical contacts sitesbetween adjacent mitochondria were evaluated for the presence ofelectron density. The proportion of all mitochondrial contact sitesbetween organelles that were electron dense is reported as a percent-age.

SEM. For SEM, the other half of the mouse soleus was immediatelyfixed in a 1% glutaraldehyde (TAAB Lab Equipment) and 0.5%paraformaldehyde (Sigma 158127) solution in a 0.060 M cacodylate(TAAB Lab Equipment) buffer, pH 7.4. For processing, fixed sampleswere rinsed twice in 0.067 M cacodylate buffer and subsequentlyimmersed in DMSO (Sigma D2650) for 5–10 min. Then, the fixedsamples were snap frozen in liquid nitrogen-cooled isopentane (Sigma277258) for 5–10 s and immediately fractured by applying lateralpressure within a custom designed liquid nitrogen-cooled crackinginstrument. The resulting fractured specimens, �0.5–3 mm in size,were rinsed three times in 0.067 M cacodylate buffer and postfixed inosmium tetroxide (OsO4) 1% for 1 h. Samples were then transferredto OsO4 0.1% for 72–96 h to partially extract cytoplasmic components(22). Then, muscle samples were dehydrated in sequential steps ofethanol (25%, 50%, 75%, and 100% twice) and dried with CO2 in aBaltec critical point dryer. Specimens were mounted on stubs coveredwith carbon disks, gold coated (15 nm) in Polaron SEM coating unit,and examined on a Stereoscan 240 scanning electron microscope.Fracture sites were observed for exposed mitochondria, and digitalmicrographs were captured at different magnifications with the Orion6.60.6 software.

Morphological analysis and statistics. Mitochondrial shape de-scriptors and size measurements were obtained using Image J (version1.42q, National Institutes of Health, Bethesda, MD) by manuallytracing only clearly discernible outlines of SS and IMF mitochondriaon TEM micrographs, as shown in Fig. 5. Mitochondria coexisting inboth SS and IMF compartments (representing less than 1% of total)were excluded from these analyses. Surface area (mitochondrial size)is reported in squared micrometers; perimeter in micrometers; aspectratio (AR) is computed as [(major axis)/(minor axis)] and reflects the

Table 1. Detailed morphological parameters for SS and IMFmitochondria in mouse soleus

Median Mean (SD) Min Max Skewness

Surface area, �m2

SS 0.19 0.28 (0.31) 0.01 2.72 3.33IMF 0.22 0.27 (0.20) 0.02 1.39 1.67

Roundness (0–1)SS 0.57 0.57 (0.18) 0.14 1 �0.01IMF 0.33 0.37 (0.20)* 0.08 1 0.78

Circularity (0–1)SS 0.80 0.75 (0.16) 0.22 0.97 �0.94IMF 0.41 0.45 (0.22)* 0.07 0.97 0.46

Feret’s Diameter, �mSS 0.69 0.79 (0.48) 0.08 3.8 1.84IMF 1.06 1.20 (0.68)* 0.18 5.49 1.37

Perimeter, �mSS 1.77 2.05 (1.20) 0.22 8.21 1.71IMF 2.54 3.00 (1.84)* 0.48 16.06 1.78

Form factorSS 1.26 1.41 (0.45) 0.97 4.55 2.60IMF 2.44 2.89 (1.76)* 0.92 14.77 2.05

Aspect ratioSS 1.75 1.97 (0.83) 1 7.18 2.16IMF 3.03 3.63 (2.13)* 1 13.16 1.26

Skewness is a measure of asymmetry in distributions. A value of 0 �normally or randomly distributed population; a value �0 � more smallervalues than expected; and a value �0 � more larger values than expected. SeeFig. 6B-for a visual representation of distributions and their skewness. *Inter-myofibrillar (IMF) significantly different (P � 0.01) from subsarcolemmal(SS) mitochondria, based on 99% confidence interval of the mean. n � 505 SSand 563 IMF mitochondria.

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“length-to-width ratio”; form factor (FF) [(perimeter2)/(4·surfacearea)] reflects the complexity and branching aspect of mitochondria;circularity [4·(surface area/perimeter2)] and roundness [4·(surfacearea)/(·major axis2)] are two-dimensional indexes of sphericity withvalues of 1 indicating perfect spheroids; and Feret’s diameter repre-sents the longest distance (�m) between any two points within a givenmitochondrion (18). Computed values were imported into MicrosoftExcel and Prism 5 (GraphPad Software) for data analysis. Statisticalsignificance was evaluated based on 99% confidence interval (CI) ofthe mean. To produce frequency distributions of each morphologicalparameter, each mitochondrion was assigned to one of 20 bins ofequal sizes and proportions were determined, yielding frequencyhistograms and level of skewness. A summary of the major statisticaltests for each morphological parameter in SS and IMF mitochondriais presented in Table 1.

RESULTS

Skeletal muscle mitochondria and outer membrane interactions.We studied the morphology of the two distinct mitochondrialpopulations in mouse skeletal muscle: subsarcolemmal (SS)and intermyofibrillar (IMF) mitochondria. SS mitochondriareside between the sarcolemma and myofibrils, whereas IMFmitochondria are located between myofibrils (21). The combi-

nation of freeze-fractured myofibers imaged with SEM (Fig. 1,A and C) and resin-embedded ultrathin sections imaged withTEM (Fig. 1, B and D) revealed that mitochondrial morphol-ogy in skeletal muscle fibers is highly diverse.

To determine whether mitochondrial membranes interact invivo, mitochondria were examined at high resolution. Theclassical dyad of IMF mitochondria across the Z-line wascommonly observed (Fig. 2A), although continuous or inter-acting mitochondria across the Z-line were observed in 12.5%of cases within mitochondria-rich myofiber segments (Fig. 2, Band C). The outer mitochondrial membranes of adjacent IMFmitochondria were found to interact, forming mitochondrialcontact sites. Mitochondrial contact sites were occasionallycharacterized by increased electron density (Fig. 2, C and D),likely from the presence of large electron-dense proteins. Atelectron-dense contact sites, two adjacent outer mitochondrialmembranes came into close juxtaposition to become indistin-guishable (Fig. 2, C’ and D’). Electron densities were presentat contact sites with similar prevalence among SS and IMFmitochondria, consisting of 8.7% and 7.5% of all contact sites,respectively. Irregularly shaped mitochondria with distinct in-ner mitochondrial membranes surrounded by continuous outer

Fig. 1. Subsarcolemmal and intermyofibrillar mitochondrial morphology in intact mouse soleus muscle fibers. Scanning electron microscopy (EM) of afreeze-fractured muscle sample (A) and transmission EM in the longitudinal plane (B), showing a myofiber’s outer surface lined with collagen fibers formingthe extracellular matrix. A breach within the plasma membrane exposes globular subsarcolemmal (SS) mitochondria in A, which can be observed in B betweenthe plasma membrane and myofibrils. Scanning EM of a freeze-fractured myofiber (C) and transmission EM in the transverse plane (D) exposing elongatedintermyofibrillar (IMF) mitochondria clustering around Z-lines between sarcomeres. Insets represent lower magnification micrographs with regions magnified inthe main panels. PM, plasma membrane; Myofibr, myofibrils; S, sarcomeres; Cap, capillary. Scanning electron microscopy (SEM) images were captured atmagnifications of �10,300 (A) and �1,140 (A, inset), and �5,800 (B) and �1,310 (C, inset).

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Fig. 2. Interactions among intermyofibrillarmitochondrial membranes. Transmission EMof myofibers in the longitudinal plane show-ing distinct mitochondria on either sides ofthe Z-line (A) or continuous mitochondriaspanning the Z-lines (B and C; arrowheads).C and D: mitochondrial contact sites charac-terized by electron densities localized at theouter mitochondrial membranes. C’ and D’:higher magnifications demonstrating elec-tron-dense outer membranes of adjacent mi-tochondria that become indistinguishable atthe contact sites. The arrowhead in C’ indicatesseparation of the outer and inner mitochondrialmembranes suggestive of forces acting upon theouter membrane. E: an irregularly shaped mito-chondrion with distinct matrix spaces bound by(E’) distinct inner mitochondrial membranes (ar-rows), but surrounded by a common outer mito-chondrial membrane (arrowhead). OMM, outermitochondrial membrane; IMM, inner mitochon-drial membrane; MA, mitochondrial matrix.



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membranes were also observed (Fig. 2E), possibly representingmidpoint events of incomplete mitochondrial fusion events inwhich the outer mitochondrial membranes are known to fusefirst, followed by the inner membranes (34). Electron-densecontact sites of varying aspects were observed among bothIMF (Fig. 3A) and SS (Fig. 3B) subpopulations.

Interactions between SS and IMF mitochondria (16, 17)were also investigated. Although SS and IMF mitochondriawere generally distinct groups with distinct morphology (Fig. 4A),we observed continuous organelles coexisting in both compart-ments (Fig. 4, B and C). These organelles were characterizedby more globular morphology in the SS compartment, whereasthey adopted more elongated morphology within the IMFspace.

Quantification of mitochondrial morphology in myofibers.Three-dimensional mitochondrial morphology was analyzedby using a combination of transmission EM in both the longi-tudinal and transverse planes (Fig. 5A). Whereas SS mitochon-dria are mostly spherical (see Fig. 1A) and can be visualizedwith similarly accurate representation in either planes, IMFmitochondrial morphology differs extensively depending onthe plane selected. Whereas the longitudinal plane presentedIMF mitochondria as more or less spheroid structures posi-tioned on either sides of the Z-lines (Fig. 5B), microscopicanalysis of the transverse plane (Fig. 5C) revealed the mito-chondrial reticulum (4, 17). During freeze fracture, myofiberswere ruptured at regular intervals in a “staircase” manner topartially expose sarcomeres lined on either sides by reticularmitochondria (Fig. 5D and Fig. 1C).

To quantify mitochondrial morphology, mitochondria wereindividually traced from transmission EM. The “form factor”/“aspect ratio” distribution among SS and IMF mitochondriadiffered markedly (Fig. 6, A and B). Further analysis of themajor mitochondrial shape descriptors indicated that distribu-tion of roundness, circularity, Feret’s diameter, perimeter, formfactor, and aspect ratio differed significantly between SS andIMF mitochondria (Fig. 7 and Table 1). For instance, circularmitochondria (roundness �0.8) constituted 48.5% of SS mito-chondria, but represented only 8.9% of IMF mitochondria. Incontrast, mitochondrial size was relatively similar between SSand IMF mitochondria (Table 1). Furthermore, the frequencydistributions for mitochondrial size (Fig. 7B) and several mor-phological parameters (Fig. 7, C–H) were highly (�1.0)skewed (Table 1), suggesting the presence of mechanisms toactively preserve small mitochondrial sizes and specific mor-phology.

DISCUSSION

The existence of mitochondrial dynamics in intact skeletalmuscle fibers largely remains to be established. Likewise,although mitochondrial morphology is known to be diverse inskeletal muscle fibers (4, 22), the extent to which SS and IMFmitochondria differ is underappreciated and no prior study hassystematically quantified normal parameters of mitochondrialmorphology in both subpopulations. In this study, we reportand quantify the extent to which outer mitochondrial mem-branes of adjacent organelles interact in mouse soleus muscle,forming electron dense contact sites [or junction sites (3, 4)].Furthermore, using a combination of scanning and transmis-sion EM in two orthogonal planes, we define for the first timedistributions of morphological parameters among SS and IMFmitochondria, suggesting that mitochondrial morphology is notrandomly distributed and therefore likely to be actively regu-lated.

Mitochondrial dynamics. Mitochondrial dynamics—or theability of mitochondria to undergo fusion and fission processes—has emerged as a fundamental biological mechanism essentialto mitochondrial function and cellular health (6, 39). Followingthe identification of the proteins involved in mitochondrialmorphology transitions in mammals—mitofusins 1 and 2(Mfn1, Mfn2) and optic atrophy 1 (OPA1) for fusion; dy-namin-related protein 1 (DRP1), mitochondrial fission factor(Mff) and fission protein 1 (Fis1) for fission—molecular ge-netic defects in some of these proteins have been shown tocause neuromuscular pathology in mouse and in human (8, 42).These findings indicated that disrupting mitochondrial dynam-ics in vivo causes neuromuscular pathology. The prevailinginterpretation of these results has been that mitochondria musttherefore normally undergo regular fusion events (and fission)in myofibers in vivo. However, mitochondrial fusion proteinsalso perform other physiological function (e.g., tether mito-chondria with endoplasmic reticulum for Ca2 dynamics, tran-scriptional regulation of respiratory chain genes) that are inde-pendent of mitochondrial morphology transitions (9, 10, 29).Thus the pathological manifestations of altered fusion andfission proteins could arise independently from abnormal mi-tochondrial shape changes and should therefore not be re-garded as definite proof of mitochondrial dynamics in vivo.Except for one preliminary report demonstrating the exchange

Fig. 3. Electron dense mitochondrial contact sites among SS and IMF mito-chondria. Transmission EM showing representative electron dense mitochon-drial contact sites among subsarcolemmal (SS; A) and intermyofibrillar (IMF;B) mitochondria. Arrowheads indicate electron densities associated with mem-brane juxtaposition; the arrow indicates a restricted but continuous neckbetween two mitochondrial units.



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of a mitochondria-targeted photo-activable probe betweenneighboring skeletal muscle mitochondria (24), direct evidenceof mitochondrial fusion in mature myofibers is lacking. Estab-lishing whether mitochondrial membranes physically interactin vivo is a first step in addressing this point.

In the cytosol of cultured cells, including myoblasts [myo-genic precursor cells (2)], mitochondria continuously undergomorphological changes on the order of seconds to minutes(36). However, mitochondrial dynamics may differ, both qual-itatively and quantitatively, between proliferating culturedcells and mature differentiated muscle fibers. Unique intracel-lular features in myofibers may prevent extensive mitochon-drial dynamics, particularly the densely packed myofibrils andother organelles that populate the cytosolic space, which mayhinder free organelle movement and extensive network remod-eling as in cultured cells. In addition, the presence of solublecytosolic factors has been proposed to inhibit mitochondrialfusion in differentiated cells. The observations from the currentstudy [and previously (4)] of mitochondrial contact sites,characterized by electron densities and complex organellemorphology, suggest that mitochondrial membranes do interactin vivo. On the basis of the recent finding that adjacentmitochondria in 8-wk-old mouse skeletal muscle can exchangematrix-located molecules (24), this suggests that at least aportion of the contact sites observed could be the result ofmitochondrial fusion processes. However, mitochondrialmembrane contacts sites such as those reported herein couldbe the result of kiss-and-run behavior (20), mitochondrialdivision (although unlikely), or other processes (e.g., ion orlipid transfer) that would involve contact between adjacentmembranes (18a).

SS and IMF morphology and shape. Because of knowntopological, functional and morphological differences betweenSS and IMF mitochondria, a significant portion of our analysiscompared both mitochondrial subpopulations. Topologically,SS mitochondria are located beneath the plasma membraneoften in proximity to myonuclei and capillaries; whereas IMFmitochondria are located at the core of myofibers betweenmyofibrils (15, 21). Functionally, differences have beenobserved when these two subpopulations are mechanicallyisolated and studied, although the isolation process yieldsonly spherical organelles and may thus alter native mito-chondrial morphology and function (27, 28). Nevertheless,in preparations of isolated organelles, compared with IMF,SS mitochondria typically have lower oxidative capacity,produce lower levels of reactive oxygen species (rate ofH2O2 efflux), and are more sensitive to the proapoptoticevent of permeability transition (1, 23, 32). Morphologi-cally, consistent with previous findings (16, 17, 22), weobserved that whereas SS mitochondria were for the mostpart isolated spherical units (although relatively elongatedmitochondria were also observed), IMF mitochondria orga-nize into a reticulum interconnected by transverse mito-chondrial tubules near the Z-line (4, 17).

Elongated mitochondrial tubules have been likened to “power-transmitting cables” capable of transmitting membrane po-tential and possibly other molecules (e.g., O2, ATP) alongtheir length (33). Continuous organelles coexisting withinboth SS and IMF compartments—mitochondrial linkages(17)—might fulfill such a role, allowing the exchange ofinformation between SS mitochondria (which are in closecontact with nuclei and capillaries) and IMF mitochondria.

Fig. 4. Physical interactions between SS and IMF mitochondria. Transmission electron micrograph of myofibers in the transverse plane. A: SS and IMFmitochondria are distinct organelles. B and C: some SS and IMF mitochondria form continuous organelles (arrowheads) that coexist in both subcellularcompartments. SS, subsarcolemmal; IMF, intermyofibrillar; PM, plasma membrane (sarcolemma); Myofibr, Myofibrils; Cap, capillary.



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Another possibility to explain the existence of continuousSS-IMF mitochondrial linkages is that mitochondrial bio-genesis principally takes place within the SS space and that“newly synthesized” mitochondria thereafter migrate to theIMF region.

The most novel aspect of our study lies in the detailedcharacterization of SS and IMF mitochondrial morphology,which reinforces the notion that SS and IMF mitochondrialmorphology differ substantially and expand upon previousstudies by providing detailed analysis of size and severalmorphological parameters. The skewed distributions of mito-chondrial sizes and morphological parameters shown in Fig. 7strongly suggest the presence of factors to regulate mitochon-drial morphology. Considering mitochondrial size, if no factorcontrolled mitochondrial volume, one would expect that most

mitochondria would be of a given “optimal” size (the mean),with an equal distribution of sizes on either side to produce anormal distribution (skewness � 0, not skewed). Statistically,the random sampling of mitochondria from micrographs acrossthe thickness of myofibers would likewise yield an equaldistribution of values for sizes and morphological parameters.However, distribution of SS and IMF mitochondrial sizes werehighly skewed (3.33 and 1.67, respectively), as were severalother morphological parameters (see Table 1), indicating thatmechanisms likely exist to regulate mitochondrial size andshape. Factors involved in determining mitochondrial shapecould be passive, such as the presence of densely packedmyofibrils that limit the lateral enlargement of IMF mitochon-dria, potentially explaining their larger aspect ratio (length/width ratio) compared with SS mitochondria. The fact that SS

Fig. 5. Three-dimensional visualization of intermyofibrillar mitochondrial morphology in muscle fibers. A: schematic representation of a myofiber sectioned inthe two major planes. A combination of transmission EM in the longitudinal (B) and transverse planes (C) reveals the complexity of intermyofibrillar (IMF)mitochondria within the natural organization of sarcomeres. The Z-line is not visible in C and highly branched mitochondria are apparent due to the almost perfecttransverse orientation of the muscle fiber. D: scanning EM micrograph showing how the freeze-fracture process tends to rupture sarcomeres at the I band nearthe Z-line, where thick filaments are absent and most IMF mitochondria are located. Myofibers are composed of multiple sarcomeres arranged in series, whereeach sarcomeric plane is populated by reticular mitochondria along its transverse axis (see also Fig. 1C). Insets show B: IMF mitochondria in a zone of highmitochondrial density, note also sarcomeric shortening due to myofiber contraction. C: a minimally inclined transverse section showing an expanded Z-line (darkline) and mitochondria on either sides; D: lower magnification of the main image showing the whole-width myofiber after freeze-fracture. SEM images werecaptured at magnifications of �5,800 (D), �2,900 (D, inset). S, sarcomere; M, mitochondria.



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mitochondria, which are not constrained by myofibrils exhibitnormal distribution in roundness (skewness � �0.01) but thatIMF mitochondria exhibit significant skewness (0.78) supportsthis notion. In turn, active factors, such as selective mitochon-drial fusion and fission processes (34a, 35), or other unknownmechanisms, could actively participate in shaping nonnormaldistributions of mitochondrial sizes and morphology observedamong both SS and IMF mitochondria.

In our study, the mouse soleus was chosen because it is ofrelatively homogenous oxidative fiber-type composition (55%type I, 51% type IIa) (13) and also because it is extensivelystudied and thus well characterized biochemically and physi-ologically. Although mitochondrial topology may be preservedbetween oxidative and glycolytic fiber types (15), fiber typedifferences in mitochondrial density (particularly SS) and re-ticular morphology (17, 22) and function (26) exist and shouldbe taken into consideration when extrapolating these results toother models.

Methodological considerations. Significant obstacles to thestudy of mitochondrial morphology and mitochondrial dynam-ics through microscopy relates to space and time resolution.

Although light microscopy generally enables the imaging of alarger number of events/cells over time, conventional ap-proaches employing fluorescent molecules are limited by thenatural resolution limits of 200 nm (down to 70 nm onsuper-resolution platforms) of light microscopy, cannot dis-criminate between adjacent organelles or membranes, andtherefore do not enable accurate quantification of mitochon-drial morphology or interactions in skeletal muscle fibers. Inturn, electron microscopy allows precisely to resolve mito-chondrial membranes and to qualitatively assess interorganellecontact sites. However, EM-based approaches require tissuefixation and are thus bound to static observations of dynamicprocesses, a limitation that recent in vivo imaging techniquesemploying photo-activatable mitochondrial dyes are aiming toovercome (24). Quantitative electron microscopy analyses sim-ilar to those described here, repeated at multiple consecutivetime points through an intervention, should allow tracking ofpopulation-wide changes in mitochondrial morphology andcontact site dynamics.

Historically, the complexities of skeletal muscle mitochon-drial morphology and topology have been qualitatively de-

Fig. 6. Quantification of form factor and aspect ratio among SS and IMF mitochondria. Mitochondrial form factor and aspect ratio were quantified by manuallytracing SS (A) and IMF (B) mitochondria, and plotted against each another. Electron micrographs numbered 1–4 correspond to individual mitochondria plottedabove. Inset in A shows mean values and 95% confidence internal (CI) for form factor and aspect ratio among both SS and IMF subpopulations. *Denotesstatistical significance (P � 0.01) compared to SS. n � 505 SS and 563 IMF mitochondria.



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scribed (4, 5, 16, 17, 22), building from elegant studies per-formed 20–40 years ago involving serial sections and three-dimensional reconstructions of small portions of the mitochondrialreticulum in skeletal muscles of various species (4, 16). Chron-ologically, these observations were made over two decadesbefore mitochondrial dynamics emerged as a field of researchand thus before it became established that mitochondrial inter-actions and dynamics bear functional significance for the cell(39). The present study combined established electron micro-scopic approaches and modern tools derived from light micros-copy to quantify mitochondrial morphology and interpreted itsfindings cognizant of the molecular mechanisms and potentialfunctional consequences of mitochondrial dynamics. Althoughour results do not unequivocally establish the existence ofmitochondrial dynamics in the intact muscle, they demonstratethat mitochondrial membranes interact and assume configura-tions consistent with mitochondrial dynamics. We hope thatthis work will contribute to establish that mitochondrial mor-phology (both SS and IMF) is unique and diverse in skeletalmuscle and that systematic quantification of mitochondrialmorphology and interactions in intact myofibers can be accom-plished using standard TEM procedures and morphometricanalysis.

Summary

In conclusion, this study reveals that mitochondrial mor-phology in mature myofibers consists of two divergent but

interacting populations of mitochondria, for which we sys-tematically quantified size and morphological parameters.This study provides new evidence that mitochondrial mem-branes interact in vivo and that factors may be involved inregulating SS and IMF mitochondrial size and morphologyin the intact muscle. The molecular composition of mito-chondrial contact sites remains to be established. Likewise,future studies will be required to determine the relationshipbetween mitochondrial contact sites, potential remodeling ofSS and/or IMF mitochondrial morphology, and skeletalmuscle physiology.

ACKNOWLEDGMENTS

The authors are grateful to Tracey Davey (EM Research Services, New-castle University) for expert technical assistance for the preparation andvisualization of samples; to Christopher Huggins for assistance with animalhandling; to Heidi McBride, Orian Shirihai, Yan Burelle and Benoit Gentil forvaluable discussions; and to Gilles Gouspillou for providing critical commentson a previous version of this manuscript.

GRANTS

M. Picard was supported by a Ph.D. Canada Graduate Scholarship and aMichael Smith Foreign Study Supplement from the National Science andEngineering Research Council of Canada (NSERC), was a Canadian Instituteof Health Research (CIHR) Fellow in Psychosocial Oncology and in SystemsBiology, and is now supported by a CIHR Postdoctoral Fellowship from theInstitute of Neuroscience as part of the Canadian Epigenetics, Environmentand Health Research Consortium (CEEHRC). This work was performed in theNewcastle University Centre for Brain Ageing and Vitality supported by grantsfrom the Biotechnology and Biological Sciences Research Council (BBRC),

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Fig. 7. Analysis of morphological parameters in SS and IMF mitochondria. A: multiple shape descriptors were determined for manually traced SS and IMFmitochondria. Bars represent the range of values that contain 50% of all mitochondria (interquartile range) for each parameter. B–H: frequency distributions(%total mitochondria) were plotted for each shape descriptor with 20 bins of equal sizes. n � 505 SS and 563 IMF mitochondria.



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Engineering and Physical Sciences Research Council (EPSRC), Economic andSocial Research Council (ESRC), Medical Research Council (MRC), theWellcome Trust Centre for Mitochondrial Research and National Institute forHealth Research Newcastle Biomedical Research Centre based at Newcastleupon Tyne Hospitals National Health Service Foundation Trust and NewcastleUniversity to Douglass M. Turnbull.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: M.P. conception and design of research; M.P. andK.W. performed experiments; M.P. analyzed data; M.P. and D.M.T. inter-preted results of experiments; M.P. prepared figures; M.P. drafted manuscript;M.P., K.W., and D.M.T. edited and revised manuscript; M.P., K.W., andD.M.T. approved final version of manuscript.

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