u n i ve r s i t y o f co pe n h ag e n

Københavns Universitet

Ultrastructural myocardial changes in seven cats with spontaneous hypertrophiccardiomyopathyChristiansen, Liselotte Bruun; Prats Gavalda, Clara; Hyttel, Poul; Koch, Jørgen

Published in:Journal of Veterinary Cardiology

DOI:10.1016/j.jvc.2015.10.003

Publication date:2015

Document VersionPublisher's PDF, also known as Version of record

Citation for published version (APA):Christiansen, L. B., Prats Gavalda, C., Hyttel, P., & Koch, J. (2015). Ultrastructural myocardial changes in sevencats with spontaneous hypertrophic cardiomyopathy. Journal of Veterinary Cardiology, 17 (Suppl. 1), S220-S232. https://doi.org/10.1016/j.jvc.2015.10.003

Download date: 19. mar.. 2019

* CorrespondE-mail addr

http://dx.doi.o1760-2734/ ªcreativecommo

Journal of Veterinary Cardiology (2015) 17, S220eS232

www.elsevier.com/locate/jvc

Ultrastructural myocardial changes inseven cats with spontaneous hypertrophiccardiomyopathy

L.B. Christiansen, PhD a,b,*, C. Prats, PhD b,P. Hyttel, DVSci a, J. Koch, PhD a

aDepartment of Veterinary Clinical and Animal Sciences, Faculty of Health and MedicalSciences, University of Copenhagen, Dyrlægevej 16, DK-1870 Frederiksberg, Denmarkb Xlab, Center for Healthy Aging, Department of Biomedical Sciences, Faculty of Healthand Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen,Denmark

Received 4 November 2014; received in revised form 5 October 2015; accepted 15 October 2015

KEYWORDSCardiac ultrastructure;Electron microscopy;Metabolism;Z-disc

ing author.ess: lbc@sund.ku.dk (L

rg/10.1016/j.jvc.20152015 The Authors. Publns.org/licenses/by-nc-

Abstract Objectives: Hypertrophic cardiomyopathy (HCM) is the most commonheart disease in cats and shares clinical and pathological characteristics with hu-man HCM. Little is known about the pathogenic mechanisms underlying develop-ment of spontaneous feline HCM.Animals: The study population consisted of seven cats diagnosed with HCM andeight age-matched cats with no evidence of cardiac disease.Methods: Fresh myocardial biopsies taken from the middle of the left ventricularposterior free wall were obtained and examined with transmission electron micro-scopy.Results: Electron microscopic examination showed ultrastructural aberrations ofthe myocardial cytoarchitecture and of the interstitium in the seven cats withHCM. In the most severely affected cats the myofibrils were disorganized and sub-sarcolemmal mitochondria were depleted. In control cats, contraction band arti-facts were commonly seen.Conclusions: In this preliminary study we show that ultrastructural changes of themyocardium in seven cats with HCM involve the cytoskeleton and mitochondria.We suggest that our findings are important for future research aiming at elucidatingthe pathogenic mechanisms underlying the phenotypic expression of feline HCM.

.B. Christiansen).

.10.003ished by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://nd/4.0/).

Ultrastructure of feline hypertrophic cardiomyopathy S221

Abbreviations

2-D two-dimensional mgraphy

Ao aortaARVC arrythmogenic righ

cardiomyopathyATP adenosine triphospBW body weightCON control catsCS citrate synthaseECG electrocardiogramECM extracellular matriHCM hypertrophic cardioHW heart weightIF interfibrillarIVS interventricular sepIVSd interventricular sep

diastoleLV left ventricleLVFW left ventricular freLVFWd left ventricular free

diastoleMYBPC3 myosin binding proMYH7 b-myosin heavy-chaPN perinuclearSS subsarcolemmalTEM transmission electrT4 thyroxine

The results of this study prompt for a larger scale study, including quantitativemeasurements of mitochondrial distribution and cytoskeletal derangements in fe-line HCM.ª 2015 The Authors. Published by Elsevier B.V. This is an open access article underthe CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

ode of echocardio

t ventricular

hate

xmyopathy

tumtum measured in

e wallwall measured in

tein Cin

on microscopy

Introduction

Hypertrophic cardiomyopathy (HCM) is charac-terized by unexplained left-ventricular hyper-trophy and diastolic dysfunction. Hypertrophiccardiomyopathy is the most commonly diagnosedcardiac disease in domestic cats.1,2 The clinicaland pathological characteristics of feline HCMresemble those of HCM in humans,3,4 in which thedisease occurs with a prevalence of 0.2%.

Phenotypically, HCM exhibits marked hetero-geneity. The disease can remain subclinicalthroughout life or can lead to the development of

life-threatening complications at all ages, such ascongestive heart failure, thromboembolic eventsand even sudden cardiac death.5

Two known genetic missense mutations ingenes encoding the sarcomere protein myosinbinding protein C (MYBPC3) are associated withdevelopment of HCM in certain cat breeds.6,7 Oneis the A31P mutation in Maine Coon cats that, inhomozygous carriers, is associated with develop-ment of HCM at an early age.6,8,9 The other is theR820W that is associated with development ofHCM in Ragdoll cats.7 Mutations associated withHCM have not yet been identified in any other catbreed.

In humans, approximately 1,500 mutationsresiding in at least 11 genes are currently known tocause HCM.10 The vast majority of mutationsreside in genes encoding the sarcomere thick fila-ment proteins b-myosin heavy-chain (MYH7) andMYBPC3.11 Less frequent are mutations encodingthin filament proteins or nonsarcomere proteins,including Z-disc components and calcium-handlingproteins recognized to be involved in the devel-opment of HCM.10,12,13

The pathogenic mechanisms responsible for thedevelopment of HCM remain largely unknown.Depletion of cardiac muscle energy reserves hasbeen suggested as a unifying factor in HCM,regardless of the underlying genotype.14,15 Theheart relies on a high constant supply of adenosinetriphosphate (ATP) for contractile work of themuscle and for ion-pumping. The main producersof ATP in the cells are mitochondria. Theseorganelles take up 22%e35% of the volume densityof the myocardial cells in mammals16 to meet thecontinuous, high energetic demands.

The structural distribution of mitochondria infeline HCM has not previously been investigated. Aprevious study investigated myocyte ultrastructurein cats with HCM, but data regarding cat breed,clinical characteristics and genotype were notreported.17

The aim of the present observational study wascomparison between the myocardial ultrastructurefrom seven cats of different breeds and with var-ious phenotypic expressions of HCM with themyocardium from eight apparently healthy control

S222 L.B. Christiansen et al.

cats. According to the perception that the HCMheart is energy depleted, we hypothesized thatalterations of the cardiac mitochondria would bepresent in cats with HCM.

Animals, materials and methods

The study was approved by the Danish AnimalExperimental Inspectorate and conformed to TheEuropean Directive 2010/63/EU on the protectionof animals used for scientific purposes.

Client-owned cats were recruited through anational screening and breeding program for felineHCM, taking place at the University of Copenha-gen, Hospital for Companion Animals. Writteninformed consent was obtained from owners of allthe cats prior to their inclusion in the study.

Cats that had previously been diagnosed withfeline HCM, based on echocardiographic findingsand exclusion of other causes of ventricularhypertrophy, were recruited at the time theowners requested euthanasia of their animal.Pedigree-bred cats above 1 year of age that hadnot received any medical treatment other thancardiac medicine, contraception and dewormingagents within the last 3 months and that did nothave any apparent systemic, infectious or

Table 1 Anthropometric and echocardiographic characte

Cat # Breed Genotype Sex Age,years

LVFWd IVSd LVhypert

HCMHCM1 BSH d mc 5.7 7.05 6.79 Mode

HCM2 MCO CC mc 6.2 11.80 4.99 SevHCM3 MCO CC mc 2.2 8.17 7.95 Sev

HCM4 ESH d mc 5.9 7.17 7.40 ModeHCM5 MCO CC fc 8.9 6.80 5.50 MiHCM6 BSH d mc 5.5 6.04 6.56 ModeHCM7 NFO d mc 8.7 7.87 7.14 Mi

ControlCON1 MCO GG fc 7.1 4.00 4.10 dCON2 MCO GG mc 2.4 3.92 3.95 dCON3 MCO GG f 3.8 4.53 4.07 dCON4 MCO GG mc 2.9 4.40 4.46 dCON5 MCO GG f 3.3 3.97 3.93 dCON6 NFO d f 11.0 4.32 3.9 dCON7 NFO d mc 7.8 4.43 4.62CON8 NFO d mc 7.5 3.46 4.31

MCO, Maine Coon cat; NFO, Norwegian Forest cat; ESH, Exotic ShoA31P mutation in MYBPC3; GG, heterozygous (wild-type) for the Amale castrate; LVFWd, left ventricular free wall in diastole; IVSd, inweight; LVOTO, left ventricular outflow tract obstruction.

metabolic diseases were eligible for the study. Apedigree was provided by the owners of eachcat.

The majority of cats recruited in the study wereMaine Coon and Norwegian Forest cats. Thesebreeds are frequently presented for echocardio-graphic examinations prior to breeding. Among theHCM cats were two British Shorthair cats and oneExotic Shorthair cat (Table 1).

Behavioral matters and gingivitis were the pri-mary complaints by owners of control cats, butcomplaints also included abnormalities related tothe eyelids, skin, coat and skeleton. Among theHCM cats, the reasons for euthanasia were pro-gression of cardiac disease since the last visit,behavioral matters, gingivitis and nonspecificclinical signs of heart disease such as restlessnessand anorexia.

Clinical characterization

Nine cats that had previously been diagnosed withHCM (one previously categorized as equivocal) andeight age-matched cats, eligible for inclusion inthe control group, had physical examination, bloodpressure measurement, electrocardiogram echo-cardiography, routine serum biochemistry, hema-tology, and total thyroxine measurements

ristics of the cats.

rophyLA:Ao Shortening

fraction, %HW/BW Other

rate 2.8 56 0.51 Arterialthromboembolism

ere 2.0 47 0.56 LVOTOere 3.4 51 0.57 Congestive heart

failure, LVOTOrate 1.1 48 0.51 LVOTOld 1.3 41 0.34 LVOTOrate 2.3 38 0.47 LVOTOld 1.8 50 0.42 Atrial fibrillation,

LVOTO

1.2 44 0.350.9 28 0.351.0 41 0.321.1 57 0.311.3 45 0.281.1 29 0.291.2 39 0.371.1 21 0.26

rthair cat; BSH, British Shorthair cat; CC, homozygous for the31P mutation in MYBPC3; f, female; fc, female castrate; mc,terventricular septum in diastole, HW, heart weight; BW, body

c CM 100 BioTWIN, Philips, Eindhoven, The Netherlands.d Image J.47 NIH, Bethesda, MD, USA.e Adobe Photoshop CC 2014, Adobe Systems Software Ireland

Ultrastructure of feline hypertrophic cardiomyopathy S223

performed. Cats of the breed Maine Coon weregenetically screened for the A31P mutation inMYBPC3.6

A diagnosis of feline HCM was based on echo-cardiographic assessment of diastolic thickness ofthe left ventricular free wall (LVFWd) and theinterventricular septum (IVSd), as previouslydescribed.18 In brief, cats were classified as HCM-positive when the maximal diastolic thickness ofthe LVFWd, IVSd or both exceeded 5.5 mm andthere were no indications of other causes of leftventricular hypertrophy on the clinical examina-tion, blood tests and blood pressure measure-ments. Cats that were equivocal for HCM, i.e., amaximum LVFWd of 5.0e5.5 mm, were excludedfrom the study.

Tissue sampling and preparation

In HCM cats and control cats, euthanasia wasinduced with an intramuscular injection of dex-medetomidine, followed by an intravenous injec-tion of pentobarbitone (150 mg/kg). Immediatelyafter cardiac arrest, the heart was rapidly col-lected through a left lateral thoracotomy, and thewet weight recorded. One to three myocardialsamples from the posterior LVFW, proximal to thepapillary muscle, were obtained and cut intoz1 mm3 pieces. The remaining part of the LVFWwas used in two other studies, investigating themyocardial cell bioenergetics in the normal felineheart19 and in feline HCM.20

The biopsy samples were immediately trans-ferred into freshly prepared 3% glutaraldehyde in0.1 M Na-phosphate buffer (pH 7.2e7.4), fixed at4 �C for 90 min and washed in phosphate buffer.Postfixation was performed in 1% osmium tetroxidefor 60 min in the same buffer, followed by dehy-dration in a graded series of ethanol and embed-ding in Epon, as previously described.21

Semithin sections (1e2 mm) were cut andstained with toluidine blue. Regions of interestwere selected via a light microscope andphotographed on the basis of the presence oflongitudinally oriented myofibers. The diameter of3e5 longitudinally oriented, adjacent myofiberswere measured on the light microscopic images.

The biopsy samples from two HCM cats con-tained only transverse fibers as evaluated by lightmicroscopy and were excluded from furtheranalysis.

Ultrathin sections (50e70 nm) of the myocar-dium were mounted on formvar-coated slot grids(2�1 mm) and contrasted, according to standardprocedures.21

Transmission electron microscopy

Sections from two biopsies were analyzed fromeach cat. Transmission electron microscopy (TEM)was performedc at the University of Copenhagen,Core Facilities for Integrated Microscopy.

One person who was not blinded to the diagnosisof the cats performed the photographing andanalyses of the myocardial biopsies from all thecats. Initially, the whole specimen, expandingapproximately 0.1 mm2, was inspected at 1870�magnification to get an overall impression of thesample quality and to evaluate the specific ultra-structural structures and abnormalities. Intra-cellular and extracellular structures werephotographed at magnifications between 1870�and 46000�.

Then, from three different cardiac cells in eachspecimen, the following cellular compartmentswere photographed at 5800� and at 9700� mag-nifications, respectively, in a nonrandom fashion:(1) the lateral border of adjacent cardiomyocytes,including myofibrils, interfibrillar (IF) mitochon-dria, subsarcolemmal (SS) mitochondria, sarco-lemma and extracellular space, (2) theintercalated disc, delineating two cardiomyocyteswith surrounding myofibrillar strands and IF mito-chondria and (3) the nucleus of a cardiomyocytewith its detached perinuclear (PN) mitochondriaand surrounding tissue.

On the images captured at 5800�magnification,the maximum diameter of the cardiac cell nucleiwas measured. Each of the mitochondrial sub-populations was investigated on the images. Onelectron micrographs at 5800� magnification, theminimum and maximum mitochondrial diametersof IF and PN mitochondria were measured. At9700� magnification, the specific ultrastructure ofthe mitochondria was assessed.

Measurements of distances and image process-ing were performed using image processingsoftwared,e.

Statistics

For comparison of the continuous variablesdescribing anthropometric and clinical data (age,body weight [BW], heart weight (HW), HW/BWratio), an un-paired Student’s t-test was used with

Ltd., Dublin, Ireland.

Fig. 1 Myocardial, intercalated disc and perinuclear mitochondrial ultrastructural organization. (A) In this controlcat (CON3), the myofibrils are aligned in parallel rows. The intercalated disc is regularly arranged transversely acrossthe cardiomyocyte (outlined by arrows). IFM, interfibrillar mitochondria. (B) In this cat with mild hypertrophy of theleft ventricle (HCM7), the myofibrils are disorganized and the Z-lines are irregular. The ultrastructure of the inter-calated disc is disrupted showing an irregular shape (outlined by arrows). (C) Myocardium from a cat with severehypertrophy of the left ventricle (HCM2) showing myofibrillar disorganization, clumping of the IFM and marked dis-ruption of the ultrastructure of the intercalated disc showing severe disorganization and interdigitation (outlined byarrows). (D) Perinuclear mitochondria at the pole of a cardiomyocyte nucleus (N) from the control cat (CON3) shownin panel A. The mitochondria are tightly packed and of varying sizes. (E) Perinuclear mitochondria from the same HCMcat (HCM7) as shown in panel B. The mitochondrial ultrastructure is preserved but the organelles are less denselypacked and are of varying sizes. N, nucleus. (F) Perinuclear mitochondria from the same HCM cat (HCM2) as shown inpanel C. There is variation in their size but mitochondrial ultrastructure is preserved.

S224 L.B. Christiansen et al.

statistical significance set at p<0.05. Data wereanalyzed using commercial statistics softwaref

Results

Baseline characteristics and echocardiographicmeasures of the seven HCM cats (six male/onefemale) and eight control cats (four male/fourfemale) included in the study are presented inTable 1.

In the control group, two Maine Coon cats (CON3 and CON 4) and two Norwegian Forest cats (CON6and CON8), respectively, were parents and off-spring. Among the HCM cats, none were close rel-atives i.e., brothers and sisters, parents andoffspring, neither to each other nor to any of thecontrol cats.

f GraphPad Prism, GraphPad Software, La Jolla, CA, USA.

Hypertrophic cardiomyopathy cats and controlswere not significantly different regarding age andbody weight. The heart weight to BW ratio wassignificantly higher in HCM cats (4.5 � 0.3) than incontrols (3.3 � 0.1; p<0.001).

All HCM cats had diffuse, asymmetrical hyper-trophy of the left ventricle. The LVFW was hyper-trophied in all the cats (Table 1). Five out of theseven cats had additional hypertrophy of the IVS(Table 1).

Left ventricular hypertrophy was arbitrarilycategorized as mild, moderate or severe22 and theleft atrium was considered enlarged when the left-atrium-to-aorta ratio >1.5 (Table 1). None of thecats had decreased fractional shortening, indicat-ing that the systolic function was preserved (Table1). Four HCM cats received medical treatmentconsisting of an angiotensin converting enzymeinhibitor (benazepril, 0.4 mg/kg BW once daily orramipril, 0.2 mg/kg BW once daily) as eithermonotherapy or in combination with a b-

Ultrastructure of feline hypertrophic cardiomyopathy S225

adrenoceptoreblocking agent (atenolol,6.25e12.5 mg/cat once daily).

Myocardial ultrastructure

The ultrastructural findings of the myocardiumfrom control cats were consistent with existingliterature of normal feline myocardial ultra-structure.23 The IF, PN and SS mitochondria wereidentified in the specimens from all cats. AlthoughPN mitochondria are often functionally regardedas IF mitochondria,24e26 they are here described asa separate subpopulation because of their distinctlocalization adjacent to the nucleus.23,27,28

In the control cats, myofibrils were orderlyorganized in parallel rows with IF mitochondriaarranged in rows between them (Fig. 1A). Theintercalated discs had a regular structure, con-sisting of small pleats and with their transversesections running perpendicular to the long axis ofthe myofibrils (Fig. 1A).

In HCM cats, disorganization of the myofibrilswas evident. The ultrastructure of the intercalateddiscs was disrupted as seen by moderate andsevere irregular folding and interdigitation (Fig. 1Band C).

Interfibrillar mitochondria were often organizedinto clusters (Fig. 1C) rather than being dispersedin regular rows between the myofibrils. In otherareas, the mitochondria were replaced by fibroustissue. There was no apparent change in theoverall content of IF mitochondria.

In two cats with mild to moderate hypertrophy(HCM4 and HCM7), the normal ultrastructure of thecardiomyocytes was largely preserved. On thecontrary, one cat with only mild hypertrophy onechocardiography (HCM5) had severe

Fig. 2 Electron-dense rods in three HCM cats. (A) Electron-hypertrophy (HCM5). There is significant disorganization of tmoderate hypertrophy (HCM4) and significant accumulationshowing presence of ERs, myofibrillar disorganization, irregu

ultrastructural changes of the myocardium withmarked myofibrillar disorganization. Noticeably,this cat was a Maine Coon cat, homozygous for themutation in MYBPC3, and therefore at high risk ofdeveloping severe HCM.4,8,9 In the remaining fourHCM cats, diffuse myofibrillar disorganization andmyofiber disarray were present.

In each of the seven cats with HCM, the diam-eters of IF mitochondria were similar to the lengthof the adjacent sarcomere, typically approx-imately 1.0 mm and always less than 2.0 mm.

Contraction band artifacts29,30 were commonlyfound in the myofibers of control cats and less so incats with HCM. In those cardiomyocytes wherecontraction band artifacts were abundant, mito-chondria were packed tightly and were elongatedin the transverse direction.

The PN mitochondria were characterized aspleomorphic in control cats and HCM cats. Similardiameters of PN were seen in the two groups (CON:0.2e1.9 mm vs. HCM: 0.2e1.8 mm). In the controlcats, PN mitochondria were placed in denselypacked clusters at the nuclear poles (Fig. 1D). Thisstructure was largely preserved in the HCM cats,although in some cats, cytoplasms surrounding thePN mitochondria seemed to be more abundant(Fig. 1E). The normal dense cristae, formed by theinner mitochondrial membranes, were preservedin the mitochondria of the HCM cats (Fig. 1E andF).

Ultrastructural organization of Z-discs

A common finding in cats with HCM was elongated,electron-dense rods extending from the Z-discs(Fig. 2). We did not find these structures in any ofthe control cats, although their presence has been

dense rods (ERs) in the myocardium from a cat with mildhe myofibrils and Z-discs are irregular. (B) HCM cat withof ERs. (C) HCM cat with severe hypertrophy (HCM3),lar Z-discs and distended T-tubules (arrows).

S226 L.B. Christiansen et al.

reported in aging cats.31 Electron-dense rods wereabundant in cats regardless of the severity ofhypertrophy (Fig. 2AeC).

Irregular and disrupted Z-discs were abundant inthe disorganized myofibrils of cats with HCM(Fig. 2A and C).

Sarcoplasma ultrastructural alterations

In the normal feline myocardium, the sarcolemmadisplays a scalloping appearance, with deep

Fig. 3 Ultrastructural alterations of themyocardial sarcolemnormal hollow-crest structure of the sarcolemma and normalone mitochondria for each sarcomere in a control cat (CON3).the same cat (CON3) as shown in panel A. The laminar coat is spthe sarcolemma at the costameres (black arrows). Omega-shlemma (white arrowheads). (CeH) Sarcolemmal and subsarmarked disruption of the sarcolemmal ultrastructure causinthickness of the laminar coat is increased (black arrows) resultdiscs (Z). Normal ultrastructure of SSM is absent and only rempresent (white arrows). Along the sarcolemma there are depo(C and D) HCM cat (HCM6) with moderate hypertrophy of thhypertrophy of the left ventricle. (G and H) HCM cat (HCM3)panels show high magnification of the left panels. Z marks th

invaginations located at the Z-discs.23 Sub-sarcolemmal mitochondria are located beneaththe sarcolemma, often with one mitochondrion foreach sarcomere (Fig. 3A and B). Omega-shapedcaveolae are present, randomly distributed alongthe sarcolemma and this was seen in the controlcats (Fig. 3B).

In cats with mild hypertrophy, the scallopingappearance of the sarcolemma was preserved butclumping of SS mitochondria was observed in somecells. In cats with moderate to severe LV

mawith feline HCM. (A) Photomicrograph illustrating theorganization of subsarcolemmal mitochondria (SSM) withZ, Z-disc. (B) High magnification of the sarcolemma fromarse and there is close proximity between the Z-discs andaped caveolae are randomly distributed along the sarco-colemmal structures aberrations in HCM cats. There isg the caveolae to be distorted (white arrowheads). Theing in a loss of contact between the sarcolemmaand the Z-nants of mitochondria in the subsarcolemmal space are

sits of electron dense Z-disc material (black arrowheads).e left ventricle. (E and F) HCM cat (HCM2) with severewith severe hypertrophy of the left ventricle. The righte identical Z-disc in the left and right panels.

Ultrastructure of feline hypertrophic cardiomyopathy S227

hypertrophy, there were areas with flattened sar-colemma, lining of the sarcolemma with electron-dense Z-disc material, and depletion and absenceof SS mitochondria (Fig. 3CeH). In cats with severehypertrophy, the sarcolemmal ultrastructure wasobserved to be severely altered compared tocontrol cats. Alterations included loss of the nor-mal hollow crest structure, presence of membranebound vacuoles, and disruption of the caveolae.

Fig. 4 Various ultrastructural findings in the myocardium fin an HCM cat (HCM2). (B) High magnification of the nucleogranules found at the nuclear pole in this HCM cat (HCM3). (Din C. (E) A filamentous body (FB) was present in a cardiomyHigh magnification of the FB shown in F. (G) Myofibrillar degeis absence of SS mitochondria, a thickened laminar coat anheads) in this cell. (H) High magnification of myofibrillar degsame location in panels G and H.

Moreover, depletion and absence of the SS mito-chondria became evident (Fig. 3CeH).

Other structures associated with HCM

The nuclei of cardiomyocytes were irregularlyshaped and often contained prominent nucleoli(Fig. 4A and B), as previously described in heartsfrom both normal and HCM cats.17,23 The maximum

rom HCM cats. (A) Nucleus (N) with prominent nucleoluslus from panel A. (C) Moderate amount of lipofuscin (Li)) High magnification of the lipofuscin granules (Li) shownocyte from this cat (HCM7) with severe hypertrophy. (F)neration in a cat (HCM 3) with severe hypertrophy. Thered deposition of electron-dense Z-disc material (arrow-enerative changes shown in panel G. Asterisks mark the

S228 L.B. Christiansen et al.

diameter of the nucleus was similar in HCM cats(7.5 � 0.5 mm) and control cats (7.0 � 1.4 mm;p¼0.3). This in accordance with a recent lightmicroscopic study.32

Scattered among the PN mitochondria werelipofuscin granules, found in controls and in HCMcats in equal amounts (Fig. 4C and D). In a catwith severe hypertrophy, an inclusion body21.0 mm long and 4.5 mm wide was presentbetween two myofibrils. The content of thisstructure was filamentous amorphous material,consistent with degenerated myofibrils (Fig. 4Eand F). Focal areas of myofiber degenerationwere occasionally seen in cats with severehypertrophy (Fig. 4G and H).

Interstitium ultrastructure

The laminar coat, consisting of a micro-threadedelectron-dense extracellular matrix (ECM) wassparse in the control cats (Fig. 3A and B). Smallamounts of type III collagen were recognized in theinterstitium as characteristic electron-dense fibrilswith a diameter of approximately 120 nm. Each ofthe seven HCM cats had some degree of interstitialremodeling, characterized by endomysial fibrosis(Figs. 3CeH and 5A and B). A common finding incats with HCM was increased thickness of the

Fig. 5 Interstitium changes in HCM cats. (A) A thick bundspace of this HCM cat (HCM6). The lower panel (B) shows hmyocardium in this HCM cat (HCM3), showing accumulatistructures consistent with T-tubuli. The lower panel (D) showithin the tubular structures. (E) Excessive amounts of the eultrastructure in this homozygous Maine Coon cat with mild hECM interdigitating with projections of the cardiomyocyte. Apanels.

laminar coat (Fig. 3CeH, 5E and F) and bundles ofcollagen fibrils in the intercellular space (Fig. 5Aand B).

Extracellular matrix, identical to that formingthe laminar coat, was found within tubular struc-tures that were consistent with T-tubuli, causingthem to be dilated and distorted (Fig. 5C and D). Infour of the seven HCM cats (HCM2eHCM5), the ECMreplaced major areas of the myocardium byforming interdigitating sheets between the myofi-brils (Fig. 5E and F).

Discussion

We here present the findings of an electronmicroscopic study of fresh myocardial biopsiesobtained from seven purebred cats with variousphenotypic and genotypic expressions of sponta-neously occurring HCM.

The following changes of myocardial ultra-structure were prominent among the HCM cats:remodeling of the myofibrils and IF mitochondria,changes of Z-disc morphology, sarcolemmalremodeling with depletion of the SS mitochon-dria, and endomysial fibrosis. In severely affec-ted HCM cats, myofibrillar degeneration waspresent.

le of collagen fibrils (CFs) is present in the extracellularigh magnification of the CFs. (C) Oblique section of theon of electron-dense micro-thread matrix localized tows high magnification of tubular organization of matrixxtracellular matrix (ECM) replace the normal myocardialypertrophy (HCM5). The lower panel (F) shows sheets ofsterisks mark the same location in the upper and lower

Ultrastructure of feline hypertrophic cardiomyopathy S229

To the best of our knowledge there are noprevious reports on the mitochondrial ultra-structure in spontaneous feline HCM. We expec-ted to find a decrease in the mitochondrialcontent in HCM hearts, following our recentfinding20 of decreased enzymatic activity of cit-rate synthase (CS), which is a marker of mito-chondrial content.33

Previous studies assessing mitochondrial volumedensity in conditions causing cardiac hypertrophyhave revealed controversial findings, showingeither increased34e36 or decreased37e39 mitochon-drial content. A transient increase in mitochon-drial volume density followed by a decrease hasalso been reported with progression of cardiachypertrophy.40,41

The presence of giant IF mitochondria expand-ing the length of several sarcomeres has beenreported with various aquired and congenitalforms of cardiomyopathy.42e44

In the seven cats with HCM, the overall densityof cardiac IF and PN mitochondria was apparentlypreserved, although the spatial organization of themitochondria was modified when myofibrils weredisorganized. In cats with moderate and severehypertrophy included in our study, it was a char-acteristic finding that SS mitochondria from the SScellular compartment were depleted. Despite thelack of quantitative measurements of mitochon-drial density in this preliminary study we suspectdepletion of the SS mitochondria to be responsiblefor an overall decrease in mitochondrial content infeline HCM.

Controversy exists as to whether IF mitochon-dria and SS mitochondria are two spatially distinctsubpopulations in muscle.46 There is substantialevidence showing that SS mitochondria and IFmitochondria have different functional bio-chemical properties in the mammalian myocar-dium.24,25,27 In animal models of cardiac disease,the SS mitochondria and IF mitochondria havebeen shown to be unequally susceptible toinjury.26,47,48 Our finding of SS mitochondrialdepletion in late stage feline HCM supports thishypothesis. Future studies, using advanced imag-ing modalities to differentiate between mito-chondrial subpopulations,46 will be highly relevantto elucidate this matter.

Expansion of the ECM was profound in cats withHCM compared to control cats, regardless of thedegree of hypertrophy. Excessive ECM formation inheart muscle can act as a substrate for thedevelopment of cardiac arrhythmias.49 Function-ally, T-tubules are responsible for calciumhomeostasis and for controlling excitation-contraction coupling in muscle cells. Dysfunction

of the T-tubular system can compromise cardiacfunction and disturb calcium homeostasis.50 Whileonly speculative at this point, our findings ofexcessive ECM expansion in HCM hearts raise thesuspicion that ECM accumulation may be involvedin the development of arrhythmias that are knownto occur in feline HCM.51

Our finding of electron-dense rods in seven HCMcats is in accordance with a previous study wheresimilar structures were found in the hearts of catswith HCM.17

We did not find electron-dense rods in any ofthe control cats. This elicits the suspicion thatthese structures represent a pathological finding.One previous study reported electron-dense rodsin three apparently healthy cats.31 However, car-diac diseases were not excluded in these cats andtheir ages were unknown. It cannot therefore beexcluded that these cats had cardiac disease andthat electron-dense rods occur in the aging catheart. Moreover, a recent study in dogs diagnosedwith arrhythmogenic right-ventricular cardiomy-opathy found electron-dense rods in diseased dogsbut not in control dogs.52

The electron-dense rods that were visualized inHCM cats and boxer dogs with arrhythmogenicright-ventricular cardiomyopathy (ARVC) resemblenemaline rods found in human congenital skeletalmyopathies and less commonly in cardiacmuscle.53e55 Nemaline myopathy is known to arisefrom mutations in thin filament actin proteins andin proteins related to alpha-actin, nebulin, tropo-myosin and troponin-T.55,56 Z-disc proteins areimportant for cardiac muscle cell structure57 andplay essential roles in cell signaling and stresssensing.58,59

At present, the significance of our finding ofelectron-dense rods in feline HCM remains elusive.While in humans, at least seven mutations in Z-discproteins are related to development of humanHCM60 our findings may warrant further inves-tigation of Z-disc protein changes in feline HCM.

Some limitations of our study should be con-sidered. One limiting factor was the small studypopulation. Feline HCM is characterized as a het-erogeneous disease with a broad range of clinicaland pathological presentations. Analyses of myo-cardial biopsies from seven HCM cats do not coverthe morphological spectrum of feline HCM. It islikely that additional ultrastructural aberrationsexist in this disease entity. There may even bebreed specific ultrastructural characteristics whichhave been missed because of the inclusion of onlyfour breeds and a small number of cats. Opposite,we did observe prominent unifying changes such aselectron-dense rods in cats with various

S230 L.B. Christiansen et al.

phenotypic and genotypic expressions of HCM.These are important findings that may directfuture research into exploring the role of struc-tural protein disturbances in the development offeline HCM.

The observer performing the TEM was not blin-ded to the diagnosis of the cats. It must beunderlined that each specimen was scanned in itwhole area. Only morphological findings that weresignificant and widely distributed are described.

Finally, limitations of the sample quality (i.e.,low contrast between IF mitochondria and myofi-brils and presence of contraction band artifacts)prevented us from performing quantitative meas-urements of the mitochondrial density. Con-traction bands are commonly found in freshlyprepared biopsies and are not regarded as patho-logical changes.45 They were more pronounced inthe control cats than in cats with HCM and thisdifference would bias the myofibrillar-to-mito-chondrial ratio. Quantification of the mitochon-drial density and other ultrastructural changeswould indeed strengthen the presentation. Futureperspectives include investigation by the use ofstereology in a larger number of cats from breedsthat are predisposed to developing HCM.

Conclusions

The present study provides preliminary data ofmyocardial ultrastructural aberrations seen withTEM in seven cats with well-defined phenotypicalexpressions of HCM. Prominent changes of themyocardial ultrastructure include cytoarchitec-tural changes, excessive ECM formation and apresumed depletion of mitochondrial mass in theSS portion of the cardiomyocytes.

Mitochondrial structural depletion correspondswell with our previous findings of reduced mito-chondrial function and overall mass in the felineHCM heart20 and with the hypothesis that the HCMheart is energy depleted.61,62

Based on these preliminary findings, we proposecytoskeletal proteins and/or mitochondrial pro-teins to be considered of importance whensearching for disease-causing genetic mutations orgene variants involved in feline HCM.

Funding

Part of the study was funded by research grantsfrom the Hartmann Brothers’ Foundation, Den-mark, and the Fund of Scientific Studies in Com-panion Animals, Denmark.

Conflict of Interest Statement

The authors do not have any conflicts of interest todisclose.

Acknowledgments

The authors acknowledge the Core Facility forIntegrated Microscopy, Faculty of Health andMedical Sciences, University of Copenhagen. Theauthors thank Hanne Mølbak Holm for technicalassistance with samples.

References1. Ferasin L, Sturgess CP, Cannon MJ, Caney SM, Gruffydd-

Jones TJ, Wotton PR. Feline idiopathic cardiomyopathy: aretrospective study of 106 cats (1994e2001). J Feline MedSurg 2003;5:151e159.

2. Riesen SC, Kovacevic A, Lombard CW, Amberger C. Preva-lence of heart disease in symptomatic cats: an overviewfrom 1998 to 2005. Schweiz Arch Tierheilkd 2007;149:65e71.

3. Fox PR, Liu SK, Maron BJ. Echocardiographic assessment ofspontaneously occurring feline hypertrophic cardiomyop-athy. An animal model of human disease. Circulation 1995;92:2645e2651.

4. Kittleson MD, Meurs KM, Munro MJ, Kittleson JA, Liu SK,Pion PD, Towbin JA. Familial hypertrophic cardiomyopathyin Maine Coon cats: an animal model of human disease.Circulation 1999;99:3172e3180.

5. Maron BJ. Hypertrophic cardiomyopathy: a systematicreview. JAMA 2002;287:1308e1320.

6. Meurs KM, Sanchez X, David RM, Bowles NE, Towbin JA,Reiser PJ, Kittleson JA, Munro MJ, Dryburgh K, Macdonald KA,Kittleson MD. A cardiac myosin binding protein C mutation inthe Maine Coon cat with familial hypertrophic cardiomyop-athy. Hum Mol Genet 2005;14:3587e3593.

7. Meurs KM, Norgard MM, Ederer MM, Hendrix KP,Kittleson MD. A substitution mutation in the myosin bindingprotein C gene in Ragdoll hypertrophic cardiomyopathy.Genomics 2007;90:261e264.

8. Longeri M, Ferrari P, Knafelz P, Mezzelani A, Marabotti A,Milanesi L, Pertica G, Polli M, Brambilla PG, Kittleson M,Lyons LA, Porciello F. Myosin-binding protein C DNA variantsin domestic cats (A31P, A74T, R820W) and their associationwith hypertrophic cardiomyopathy. J Vet Intern Med 2013;27:275e285.

9. Godiksen MT, Granstrom S, Koch J, Christiansen M.Hypertrophic cardiomyopathy in young Maine Coon catscaused by the p.A31P cMyBP-C mutationethe clinicalsignificance of having the mutation. Acta Vet Scand 2011;53:7.

10. Maron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I,Maron MS. Hypertrophic cardiomyopathy: present andfuture, with translation into contemporary cardiovascularmedicine. J Am Coll Cardiol 2014;64:83e99.

11. Harris SP, Lyons RG, Bezold KL. In the thick of it: HCM-causing mutations in myosin binding proteins of the thickfilament. Circ Res 2011;108:751e764.

12. Marian AJ. Hypertrophic cardiomyopathy: from genetics totreatment. Eur J Clin Invest 2010;40:360e369.

Ultrastructure of feline hypertrophic cardiomyopathy S231

13. Maron BJ, Maron MS, Semsarian C. Genetics of hypertrophiccardiomyopathy after 20 years: clinical perspectives. J AmColl Cardiol 2012;60:705e715.

14. Frey N, Luedde M, Katus HA. Mechanisms of disease:hypertrophic cardiomyopathy. Nat Rev Cardiol 2012;9:91e100.

15. Ashrafian H, McKenna WJ, Watkins H. Disease pathways andnovel therapeutic targets in hypertrophic cardiomyopathy.Circ Res 2011;109:86e96.

16. Barth E, Stammler G, Speiser B, Schaper J. Ultrastructuralquantitation of mitochondria and myofilaments in cardiacmuscle from 10 different animal species including man. JMol Cell Cardiol 1992;24:669e681.

17. Van Vleet JF, Ferrans VJ, Weirich WE. Pathologic alterationsin hypertrophic and congestive cardiomyopathy of cats. AmJ Vet Res 1980;41:2037e2048.

18. Granstrom S, Nyberg Godiksen MT, Christiansen M,Pipper CB, Willesen JL, Koch J. Prevalence of hypertrophiccardiomyopathy in a cohort of British Shorthair cats inDenmark. J Vet Intern Med 2011;25:866e871.

19. Christiansen LB, Dela F, Koch J, Yokota T. Tissue-specificand substrate-specific mitochondrial bioenergetics in felinecardiac and skeletal muscles. J Vet Med Sci 2015;77:669e675.

20. Christiansen LB, Dela F, Koch J, Hansen CN, Leifsson PS,Yokota T. Impaired cardiac mitochondrial oxidative phos-phorylation and enhanced mitochondrial oxidative stress infeline hypertrophic cardiomyopathy. Am J Physiol HeartCirc Physiol 2015;308:H1237eH1247.

21. Birck MM, Saraste A, Hyttel P, Odermarsky M, Liuba P,Saukko P, Hansen AK, Pesonen E. Endothelial cell death andintimal foam cell accumulation in the coronary artery ofinfected hypercholesterolemic minipigs. J Cardiovasc TranslRes 2013;6:579e587.

22. Blass KA, Schober KE, Li X, Scansen BA, Bonagura JD. Acuteeffects of Ivabradine on dynamic obstruction of the leftventricular outflow tract in cats with preclinical hyper-trophic cardiomyopathy. J Vet Intern Med 2014;28:838e846.

23. Fawcett DW, McNutt NS. The ultrastructure of the catmyocardium. I. Ventricular papillary muscle. J Cell Biol1969;42:1e45.

24. Palmer JW, Tandler B, Hoppel CL. Biochemical propertiesof subsarcolemmal and interfibrillar mitochondriaisolated from rat cardiac muscle. J Biol Chem 1977;252:8731e8739.

25. Riva A, Tandler B, Loffredo F, Vazquez E, Hoppel C. Struc-tural differences in two biochemically defined populationsof cardiac mitochondria. Am J Physiol Heart Circ Physiol2005;289:H868eH872.

26. Hoppel CL, Tandler B, Fujioka H, Riva A. Dynamic organ-ization of mitochondria in human heart and in myocardialdisease. Int J Biochem Cell Biol 2009;41:1949e1956.

27. Piquereau J, Caffin F, Novotova M, Lemaire C, Veksler V,Garnier A, Ventura-Clapier R, Joubert F. Mitochondrialdynamics in the adult cardiomyocytes: which roles for ahighly specialized cell? Front Physiol 2013;4:102.

28. Lukyanenko V, Chikando A, Lederer WJ. Mitochondria incardiomyocyte Ca2þ signaling. Int J Biochem Cell Biol 2009;41:1957e1971.

29. Adomian GE, Laks MM, Billingham ME. The incidence andsignificance of contraction bands in endomyocardial biop-sies from normal human hearts. Am Heart J 1978;95:348e351.

30. Olmesdahl PJ, Gregory MA, Cameron EW. Ultrastructuralartefacts in biopsied normal myocardium and their rele-vance to myocardial biopsy in man. Thorax 1979;34:82e90.

31. Fawcett DW. The sporadic occurrence in cardiac muscle ofanomalous Z bands exhibiting a periodic structure sugges-tive of tropomyosin. J Cell Biol 1968;36:266e270.

32. Kershaw O, Heblinski N, Lotz F, Dirsch O, Gruber AD. Diag-nostic value of morphometry in feline hypertrophic car-diomyopathy. J Comp Pathol 2012;147:73e83.

33. Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F,Stride N, Schroder HD, Boushel R, Helge JW, Dela F, Hey-Mogensen M. Biomarkers of mitochondrial content in skel-etal muscle of healthy young human subjects. J Physiol2012;590:3349e3360.

34. Kalsi KK, Smolenski RT, Pritchard RD, Khaghani A,Seymour AM, Yacoub MH. Energetics and function of thefailing human heart with dilated or hypertrophic car-diomyopathy. Eur J Clin Invest 1999;29:469e477.

35. Schwarzer M, Schrepper A, Amorim PA, Osterholt M, Doenst T.Pressure overload differentially affects respiratory capacityin interfibrillar and subsarcolemmal mitochondria. Am JPhysiol Heart Circ Physiol 2013;304:H529eH537.

36. Quigley AF, Kapsa RM, Esmore D, Hale G, Byrne E. Mito-chondrial respiratory chain activity in idiopathic dilatedcardiomyopathy. J Card Fail 2000;6:47e55.

37. Lin CS, Sun YL, Liu CY. Structural and biochemical evidenceof mitochondrial depletion in pigs with hypertrophic car-diomyopathy. Res Vet Sci 2003;74:219e226.

38. Kindo M, Gerelli S, Bouitbir J, Charles AL, Zoll J, Hoang MT,Monassier L, Favret F, Piquard F, Geny B. Pressure overload-induced mild cardiac hypertrophy reduces left ventriculartransmural differences in mitochondrial respiratory chainactivity and increases oxidative stress. Front Physiol 2012;3:332.

39. McCutcheon LJ, Cory CR, Nowack L, Shen H, Mirsalami M,Lahucky R, Kovac L, O’Grady M, Horne R, O’Brien PJ. Res-piratory chain defect of myocardial mitochondria in idio-pathic dilated cardiomyopathy of Doberman Pinscher dogs.Can J Physiol Pharmacol 1992;70:1529e1533.

40. Scheuermann DW. The ultrastructure of cardiac muscle inhealth and disease. Micron 1993;24:47e73.

41. Breisch EA, White FC, Bloor CM. Myocardial characteristicsof pressure overload hypertrophy. A structural and func-tional study. Lab Invest 1984;51:333e342.

42. Arbustini E, Diegoli C, Fasani R, Grasso M, Morbini P,Banchieri N, Bellini O, Dal Bello B, Pilotto A, Magrini G,Campana C, Fortina P, Gavazzi A, Narula J, Vigano M.Mitochondrial DNA mutations and mitochondrial abnormal-ities in dilated cardiomyopathy. Am J Pathol 1998;153:1501e1510.

43. Tandler B, Dunlap M, Hoppel CL, Hassan M. Giant mito-chondria in a cardiomyopathic heart. Ultrastruct Pathol2002;26:177e183.

44. Guenthard J, Wyler F, Fowler B, Baumgartner R. Car-diomyopathy in respiratory chain disorders. Arch Dis Child1995;72:223e226.

45. Baandrup U, Florio RA, Roters F, Olsen EG. Electronmicroscopic investigation of endomyocardial biopsy sam-ples in hypertrophy and cardiomyopathy. A semi-quantitative study in 48 patients. Circulation 1981;63:1289e1298.

46. Dahl R, Larsen S, Dohlmann TL, Qvortrup K, Helge JW,Dela F, Prats C. Three-dimensional reconstruction of thehuman skeletal muscle mitochondrial network as a tool toassess mitochondrial content and structural organization.Acta Physiol 2014;213:145e155.

47. Hollander JM, Thapa D, Shepherd DL. Physiological andstructural differences in spatially-distinct subpopulations ofcardiac mitochondria: influence of pathologies. Am JPhysiol Heart Circ Physiol 2014;07:H1eH14.

S232 L.B. Christiansen et al.

48. Croston TL, Thapa D, Holden AA, Tveter KJ, Lewis SE,Shepherd DL, Nichols CE, Long DM, Olfert IM,Jagannathan R, Hollander JM. Functional deficiencies ofsubsarcolemmal mitochondria in the type 2 diabetic humanheart. Am J Physiol Heart Circ Physiol 2014;307:H54eH65.

49. Nguyen TP, Qu Z, Weiss JN. Cardiac fibrosis and arrhyth-mogenesis: the road to repair is paved with perils. J Mol CellCardiol 2014;70:83e91.

50. Guo A, Zhang C, Wei S, Chen B, Song LS. Emerging mecha-nisms of T-tubule remodelling in heart failure. CardiovascRes 2013;98:204e215.

51. Jackson BL, Lehmkuhl LB, Adin DB. Heart rate andarrhythmia frequency of normal cats compared to cats withasymptomatic hypertrophic cardiomyopathy. J Vet Card2014;16:215e225.

52. Oxford EM, Danko CG, Kornreich BG, Maass K, Hemsley SA,Raskolnikov D, Fox PR, Delmar M, Moise NS. Ultrastructuralchanges in cardiac myocytes from Boxer dogs witharrhythmogenic right ventricular cardiomyopathy. J VetCard 2011;13:101e113.

53. Muller-Hocker J, Schafer S, Mendel B, Lochmuller H,Pongratz D. Nemaline cardiomyopathy in a young adult: anultraimmunohistochemical study and review of the liter-ature. Ultrastruct Pathol 2000;24:407e416.

54. Mir A, Lemler M, Ramaciotti C, Blalock S, Ikemba C.Hypertrophic cardiomyopathy in a neonate associated withnemaline myopathy. Congenit Heart Dis 2012;7:E37eE41.

55. Mogensen J, Klausen IC, Pedersen AK, Egeblad H, Bross P,Kruse TA, Gregersen N, Hansen PS, Baandrup U,

Borglum AD. Alpha-cardiac actin is a novel disease gene infamilial hypertrophic cardiomyopathy. J Clin Invest 1999;103:R39eR43.

56. Wallgren-Pettersson C, Laing NG. 138th ENMC workshop:nemaline myopathy, 20e22 May 2005, Naarden, The Neth-erlands. Neuromuscul Disord 2006;16:54e60.

57. Knoll R, Buyandelger B, Lab M. The sarcomeric Z-disc and Z-discopathies. J Biomed Biotechnol 2011;2011:569628.

58. Pyle WG, Solaro RJ. At the crossroads of myocardial sig-naling: the role of Z-discs in intracellular signaling andcardiac function. Circ Res 2004;94:296e305.

59. Hoshijima M. Mechanical stress-strain sensors embedded incardiac cytoskeleton: Z disk, titin, and associated struc-tures. Am J Physiol Heart Circ Physiol 2006;290:H1313eH1325.

60. Bos JM, Ackerman MJ. Z-disc genes in hypertrophic car-diomyopathy: stretching the cardiomyopathies? J Am CollCardiol 2010;55:1136e1138.

61. Crilley JG, Boehm EA, Blair E, Rajagopalan B, Blamire AM,Styles P, McKenna WJ, Ostman-Smith I, Clarke K, Watkins H.Hypertrophic cardiomyopathy due to sarcomeric genemutations is characterized by impaired energy metabolismirrespective of the degree of hypertrophy. J Am Coll Cardiol2003;41:1776e1782.

62. Jung WI, Sieverding L, Breuer J, Hoess T, Widmaier S,Schmidt O, Bunse M, van EF, Apitz J, Lutz O, Dietze GJ. 31PNMR spectroscopy detects metabolic abnormalities inasymptomatic patients with hypertrophic cardiomyopathy.Circulation 1998;97:2536e2542.

Available online at www.sciencedirect.com

ScienceDirect