RESEARCH ARTICLE

Scaling of left ventricle cardiomyocyte ultrastructure acrossdevelopment in the kangaroo Macropus fuliginosusEdward P. Snelling1,*, David A. Taggart1, Shane K. Maloney2, Anthony P. Farrell3,4, Christopher M. Leigh5,Lyn Waterhouse6, Ruth Williams6 and Roger S. Seymour1

ABSTRACTThe heart and left ventricle of the marsupial western grey kangarooMacropus fuliginosus exhibit biphasic allometric growth, whereby anegative shift in the trajectory of cardiac growth occurs at pouch exit.In this study, we used transmission electron microscopy to examinethe scaling of left ventricle cardiomyocyte ultrastructure acrossdevelopment in the western grey kangaroo over a 190-fold bodymass range (0.355−67.5 kg). The volume-density (%) of myofibrils,mitochondria, sarcoplasmic reticuli and T-tubules increase significantlyduring in-pouch growth, such that the absolute volume (ml) of theseorganelles scales with body mass (Mb; kg) with steep hyperallometry:1.41Mb

1.38, 0.64Mb1.29, 0.066Mb

1.45 and 0.035Mb1.87, respectively.

Maturation of the left ventricle ultrastructure coincides with pouchvacation, as organelle volume-densities scale independent of bodymass across post-pouch development, such that absolute organellevolumes scale in parallel and with relatively shallow hypoallometry:4.65Mb

0.79, 1.75Mb0.77, 0.21Mb

0.79 and 0.35Mb0.79, respectively. The

steep hyperallometry of organelle volumes and volume-densitiesacross in-pouch growth is consistent with the improved contractileperformance of isolated cardiac muscle during fetal development inplacental mammals, and is probably critical in augmenting cardiacoutput to levels necessary for endothermyand independent locomotionin the young kangaroo as it prepares for pouch exit. The shallowhypoallometryoforganelle volumesduringpost-pouchgrowthsuggestsa decrease in relative cardiac requirements as body mass increases infree-roaming kangaroos,which is possiblybecause theenergy requiredforhopping is independentof speed,and thecapacity forenergystorageduring hopping could increase as the kangaroo grows.

KEY WORDS: Heart, Laplace, Marsupial, Ontogeny, Remodelling,Stereology

INTRODUCTIONThe four-chambered mammalian heart completely separates thehigh-pressure systemic system that supplies oxygenated blood to thebody from the low-pressure pulmonary system that sendsdeoxygenated blood to the lungs. By operating two cardiaccircuits at vastly different pressures, and by preventingoxygenated and deoxygenated blood from mixing, mammals are

equipped with an efficient, high-output, oxygen delivery system.The effectiveness of the mammalian heart as a pump is evident inthat the same fundamental design meets the requirements ofmammals spanning eight orders of magnitude in body mass, from a1.9 g Etruscan shrew to a 190,000 kg blue whale. Over this sizerange, volume loads on the heart vary significantly, with absolutecardiac output and whole-body metabolic rate increasingsystematically with body mass. In terrestrial mammals, pressureloads on the heart also increase with body size, as larger and,particularly, taller species support a higher vertical column of bloodabove the heart (Seymour and Blaylock, 2000; White and Seymour,2014). According to the principle of Laplace, as volume loads andpressure loads on the heart increase with body mass, so too must thethickness of the ventricular wall, ensuring that wall stress (N m−2) isconserved and the load is spread (Grande and Taylor, 1965). Thisincrease in wall thickness is ultimately realised as an increase inheart mass, which has been the subject of numerous allometricinvestigations.

An allometric analysis of heart mass (Mh) against body mass (Mb)is generally expressed in the form of a power equation, Mh=a×Mb

b,where a is the scaling coefficient (elevation of log-transformedequation) and b is the scaling exponent (slope of log-transformedequation). If b=1 then heart mass increases in direct proportion tobody mass (isometric), if b>1 then heart mass increases faster thanbody mass (positive allometry or hyperallometry), and if b<1 thenheart mass increases slower than body mass (negative allometry orhypoallometry). Phylogenetic analysis across a range of different-sized mammalian species shows that heart mass increases with bodymass more-or-less isometrically, with an exponent (b) between 0.96and 1.06 (Bishop, 1997; Brody, 1945; Holt et al., 1968; Hoppeleret al., 1984; Lindstedt and Schaeffer, 2002; Prothero, 1979; Seymourand Blaylock, 2000; Stahl, 1965). Analyses restricted to marsupialspecies also trend toward isometry, with an exponent between 0.94and 1.05 (Dawson and Needham, 1981; Dawson et al., 2003).However, the scaling coefficient (a) for marsupials is approximately30% larger than that for placental mammals, presumably owing to themarsupial heart being approximately 30% larger.

Ontogenetic analyses of heart mass at different stages ofdevelopment within a single species of mammal, in contrast,show more variable results, with exponents ranging from 0.74 to0.82 in rats (Stewart and German, 1999; von Bertalanffy andPirozynski, 1952) and 1.00 to 1.02 in humans (de Simone et al.,1998; Mühlmann, 1927; Thompson, 1942). This variation is partlydue to species-specific differences in the trajectory of heart growthafter parturition, and partly due to the narrow postnatal body massrange of placental mammals (ca. 20- to 50-fold), over which mostontogenetic analyses are restricted. The range of body mass can beincreased by incorporating both prenatal and postnatal phases ofdevelopment (ca. 600-fold in placental mammals), and the fewstudies that have done this report a biphasic scaling pattern inReceived 15 January 2015; Accepted 14 April 2015

1Department of Ecology and Environmental Science, School of BiologicalSciences, University of Adelaide, Adelaide, SA 5005, Australia. 2School ofAnatomy, Physiology and Human Biology, University of Western Australia, Crawley,WA 6009, Australia. 3Department of Zoology, University of British Columbia,Vancouver, BC, Canada V6T 1Z4. 4Faculty of Land and Food Systems, University ofBritish Columbia, Vancouver, BC, Canada V6T 1Z4. 5School of Medical Sciences,University of Adelaide, Adelaide, SA 5005, Australia. 6Adelaide Microscopy,University of Adelaide, Adelaide, SA 5005, Australia.

*Author for correspondence (edward.snelling@adelaide.edu.au)

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cardiac growth. For instance, human heart mass increases with bodymass during prenatal development with an exponent of 1.19, butfollowing parturition, heart mass increases with body mass with anexponent of 0.89 (Hirokawa, 1972). Similarly, prenatal heart massin the giraffe Giraffa camelopardalis scales with an exponent of1.03, whereas postnatal heart mass scales with a shallower exponentof 0.90 (Mitchell and Skinner, 2009). A broader ontogenetic bodymass range can also be obtained from marsupial mammal species,where neonates are highly altricial, and much of developmentoccurs within the maternal pouch (Renfree, 2006; Tyndale-Biscoeand Janssens, 1988). In a recent study, we found that heart mass inthe marsupial western grey kangaroo Macropus fuliginosus alsofollows a biphasic allometric pattern across ontogeny, scaling with arelatively steep exponent of 1.10 during in-pouch development,before switching to a shallower exponent of 0.77 during post-pouchdevelopment (Snelling et al., 2015). The biphasic allometry ofcardiac growth in the marsupial kangaroo before and after leavingthe pouch and the biphasic allometry reported for placental speciesbefore and after parturition suggest that key life history events thatinvolve a change in the animal’s behaviour, physiology and activitylevels are reflected in the scaling of the heart.Clearly, the allometry of heart mass provides important insight

into the blood supply requirements of the body at different stages ofdevelopment. However, another important factor likely tosignificantly affect the functional performance of the heart, butnot evident from simple allometry of heart mass, is the gradualmaturation of the cardiac ultrastructure during development. Whilespecies-specific differences exist in the level of maturity of themyocardium at parturition, there is nonetheless a clear increase inthe volume-density of key organelles within the cardiac muscle,much of which occurs during the fetal and early postnatal periods(Canale et al., 1986; Smolich, 1995). For instance, the volume-density of the muscle’s myofibril contractile machinery increasessignificantly across this period of development in the rat, guinea pig,hamster, rabbit and sheep (Colgan et al., 1978; Friedman, 1972;Hirakow and Gotoh, 1976, 1980; Smith and Page, 1977).Presumably to support the growing demand for ATP, the volume-density of mitochondria also increases during the fetal and earlypostnatal periods, as shown in the rat, guinea pig, hamster and rabbit(Colgan et al., 1978; Hirakow and Gotoh, 1976, 1980; Hirakowet al., 1980; Smith and Page, 1977). Similarly, the sarcoplasmicreticulum, which allows for myofibril contraction by the release ofits Ca2+ stores into the cytoplasm, increases in volume-density

during the fetal and early postnatal periods, as evident from studieson the rat, hamster and rabbit (Colgan et al., 1978; Hoerter et al.,1981; Olivetti et al., 1980). Finally, the T-tubule elements, whichfunction in the propagation of action potentials deep within thecardiomyocyte, increase in volume-density as cardiomyocytediameter increases and the diffusion distance to the interiorincreases (Hirakow and Gotoh, 1976, 1980). It is important tounderstand that the increase in the volume-density of these keyorganelles occurs in addition to the absolute increase in myocardialmass (absolute volume). The changing cardiomyocyte ultrastructureand cardiac tissue mass during ontogeny would therefore influencecardiac performance on a cellular level, and as a functioning organ.In support of this, measurements of cardiac function in rat, rabbitand sheep show maturation of the myocardial ultrastructure duringdevelopment is associated with a concomitant increase in thecontractile performance of isolated myocardium and of the organ asa whole (Anderson, 1996; Friedman, 1972; Hopkins et al., 1973;Nakanishi and Jarmakani, 1984; Romero et al., 1972).

Despite all the work done to quantify the change in cardiacultrastructure at different stages of development, a thoroughallometric analysis across key life history stages within a specieshas not yet been undertaken for any mammal. An allometricexamination is crucial because it is the only means by which theoverwhelming influence of body mass can be properly accounted(Calder, 1996). Because the changes in body mass between birthand adulthood span over two orders of magnitude in kangaroos, it ispossible to define shifts in developmental trajectories more clearlythan in placental species. For example, in our recent study, weshowed that the exponent for heart mass in the western greykangaroo decreases significantly around the time of pouch vacation(Snelling et al., 2015). In the present study, we used a subset of theseindividuals, encompassing a wide range of developmental stagesand body masses, to analyse the development of the ultrastructure ofthe left ventricle. The left ventricle is responsible for the deliveryof oxygen around the body via the systemic circuit. Given thatsignificant cardiomyocyte maturation occurs during the fetal andearly postnatal life of placental mammals, we present the hypothesisthat the volume-density of key organelles within cardiomyocytes ofthe left ventricle – the myofibrils, mitochondria, sarcoplasmicreticuli and T-tubules – increases with hyperallometry duringin-pouch development, but scales independently of body massacross post-pouch development. In accordance with the principle ofLaplace, we also hypothesise that the left ventricle’s wall-to-lumenvolume ratio, and myofibril volume-density, might scale in amanner that ensures wall stress is constant across development,which would agree with a phylogenetic study of wall stress in theleft ventricle of mammals (Seymour and Blaylock, 2000).

RESULTSAllometry of left ventricle mass and geometryWestern grey kangaroos selected for this study varied 190-fold inbody mass (0.355−67.5 kg, N=16). Detailed regression andANCOVA statistics are provided in Table 1. The left ventricleexhibits biphasic allometric growth, whereby myocardial mass (andvolume) scales hyperallometrically across pouched youngdevelopment (hereafter termed ‘in-pouch’; N=6), with anexponent of 1.18±0.05, before switching to a hypoallometrictrajectory in free-roaming young-at-foot, juvenile and adultkangaroos (hereafter ‘post-pouch’; N=10), with an exponent of0.78±0.13 (Fig. 1). The left ventricle inner and outer radii, and wallthickness, show the same biphasic scaling pattern (Table 1).Breakpoint analysis (see Materials and methods) confirms that the

List of symbols and abbreviationsMb body mass (kg)Mh heart mass (g)Mlv left ventricle mass (g)P mean arterial pressure (kPa)Ri left ventricle inner lumen radius (cm)Vcm cardiomyocyte volume of the left ventricle (ml)Vl left ventricle lumen volume (ml)Vorg organelle volume of the left ventricle (ml)Vvcm,t cardiomyocyte volume-density of the left ventricle tissue

(fraction or %)Vvmf,t myofibril volume-density of the left ventricle tissue

(fraction or %)Vvorg,cm organelle volume-density of the left ventricle

cardiomyocytes (fraction or %)Vw left ventricle wall volume (ml)σf mean fibre stress across the left ventricle wall (kPa)ρ muscle density (=1.06 g ml−1)

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in-pouch and post-pouch allometric functions separate atapproximately 5–6 kg body mass, which corresponds to the bodymass at which individuals of this species typically vacate the pouchpermanently (Dawson, 2012).

Allometry of left ventricle ultrastructureA number of conspicuous changes also occur at the cellularand subcellular levels of the left ventricle during development

(Fig. 2). During in-pouch growth, the volume-density (%) ofcardiomyocytes increases, scaling with an exponent of 0.07±0.03(Table 1), and this contributes to a rather steep exponent for thescaling of the absolute volume (ml) of cardiomyocytes, 1.25±0.06(Fig. 3A). The volume-density (%) of key components of thecardiomyocyte cell also increases to varying degrees during in-pouch development, with exponents of 0.13±0.06 for myofibrils,0.04±0.03 for mitochondria, 0.20±0.11 for sarcoplasmic reticuli

Left

vent

ricle

mas

s (g

)

B1000

100

10

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Body mass (kg)1 10 1000.11 10 1000.1

Who

le h

eart

mas

s (g

)

A1000

100

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Mh (post-pouch)�Mb0.78±0.12

Mh (in-pouch)�Mb1.13±0.04

Mlv (post-pouch)�Mb0.78±0.13

Mlv (in-pouch)�Mb1.18±0.05

Fig. 1. Scaling of heart and left ventricle mass across development in the western grey kangaroo Macropus fuliginosus. Regressions are shown for(A) whole heart massMh and (B) left ventricle massMlv, as bodymassMb increases with growth. Regressions are divided into in-pouch kangaroos (<5–6 kg bodymass, open circles, N=6) and free-roaming post-pouch kangaroos (>5–6 kg body mass, filled circles, N=10). Exponents with 95% confidence intervals arepresented. See Table 1 for full statistics.

Table 1. Allometric power equations for whole heart mass, and left ventricle mass and geometry, of in-pouch and post-pouch western greykangaroos Macropus fuliginosus

In-pouch (r2) Post-pouch (r2) ANCOVA (F1,12)*

Whole heartMass 6.17Mb

1.13±0.04 (1.00) 14.3Mb0.78±0.12 (0.96) P<0.001 (29.3)

Left ventricleMass 3.82Mb

1.18±0.05 (1.00) 9.23Mb0.78±0.13 (0.96) P<0.001 (31.0)

Wall volume 3.60Mb1.18±0.05 (1.00) 8.71Mb

0.78±0.13 (0.96) P<0.001 (31.0)Lumen volume 0.56Mb

1.12±0.24 (0.98) 1.61Mb0.72±0.19 (0.90) P<0.01 (11.0)

Inner radius 5.13Mb0.37±0.08 (0.98) 7.28Mb

0.24±0.06 (0.90) P<0.01 (11.0)Outer radius 9.60Mb

0.40±0.05 (0.99) 13.2Mb0.25±0.05 (0.95) P<0.001 (29.8)

Wall thickness 5.27Mb0.44±0.11 (0.97) 6.77Mb

0.28±0.12 (0.79) P=0.03 (5.76)CardiomyocyteVolume 2.80Mb

1.25±0.06 (1.00) 7.46Mb0.78±0.15 (0.95) P<0.001 (35.0)

Volume-density 77.8Mb0.07±0.03 (0.90) 85.7Mb

0.00±0.04 (0.01) P=0.012 (8.79)MyofibrilsVolume 1.41Mb

1.38±0.10 (1.00) 4.65Mb0.79±0.15 (0.95) P<0.001 (48.5)

Volume-density 50.3Mb0.13±0.06 (0.89) 62.3Mb

0.01±0.03 (0.02) P<0.001 (24.5)MitochondriaVolume 0.64Mb

1.29±0.08 (1.00) 1.75Mb0.77±0.17 (0.93) P<0.001 (30.5)

Volume-density 22.8Mb0.04±0.03 (0.73) 23.4Mb

−0.01±0.06 (0.02) P=0.17 (2.18)‡

Sarcoplasmic reticuliVolume 0.066Mb

1.45±0.13 (1.00) 0.21Mb0.79±0.20 (0.92) P<0.001 (36.5)

Volume-density 2.34Mb0.20±0.11 (0.85) 2.81Mb

0.01±0.09 (0.00) P<0.01 (10.8)T-tubulesVolume 0.035Mb

1.87±0.31 (0.99) 0.35Mb0.79±0.30 (0.82) P<0.001 (35.3)

Volume-density 1.24Mb0.62±0.25 (0.92) 4.74Mb

0.01±0.21 (0.00) P<0.001 (21.5)Other elementsVolume 0.59Mb

0.80±0.06 (1.00) 0.48Mb0.73±0.27 (0.83) P=0.64 (0.23)§

Volume-density 21.1Mb−0.44±0.12 (0.96) 6.49Mb

−0.05±0.32 (0.01) P=0.04 (5.31)

Also shown are the power equations for cardiomyocyte volume and cardiomyocyte volume-density within the left ventricle tissue, and for the volume and volume-density of myofibrils, mitochondria, sarcoplasmic reticuli, T-tubules and the sum of other less abundant cellular elements (see Materials and methods for list)within the cardiomyocytes.In-pouch, <5–6 kg body mass, N=6; post-pouch, >5–6 kg body mass,N=10. Body mass (Mb) is in kg, heart and left ventricle mass are in g, linear dimensions arein mm, volumes are in ml, and volume-densities are expressed as percentages.*ANCOVA statistics for in-pouch versus post-pouch exponents. ANCOVA statistics for elevations where applicable: ‡F1,13=0.90 (P=0.36) and §F1,13=1.85(P=0.20).

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Fig. 2. Electron micrographs showing cardiomyocyte ultrastructure of the developing left ventricle across ontogeny in M. fuliginosus. Lowmagnification images are presented on the left side of the panel, and high magnification on the right. (A,B) Younger in-pouch kangaroo. Nuclei occupy a largefraction of cardiomyocyte volume, and there exist pockets of cytosol where organelle architecture has not yet been synthesised. Scale bars: A, 5 µm; B, 1 µm.(C,D) Older in-pouch kangaroo. Cardiomyocyte ultrastructure is more ordered, with the myofibrils aligned in register. A prominent M-line is also present,which is regarded as an end-point of myofibrillar maturation. Scale bars: C, 5 µm; D, 2 µm. (E,F) Adult post-pouch kangaroo. Organelle volume-densities aresimilar to those of older in-pouch kangaroo. Scale bars: E, 5 µm; F, 2 µm. cm, cardiomyocyte; cp, capillary; cyt, cytosol; m,M-line; mf, myofibril; mt, mitochondrion;nu, nucleus; sr, sarcoplasmic reticulum; tt, T-tubule; z, Z-disk.

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and 0.62±0.25 for T-tubules (Table 1) (ANCOVA, F3,16=24.5,P<0.0001). The increasing volume-density of these organelleswithin the cardiomyocytes, combined with the increasing volume-density of cardiomyocytes within the left ventricle, and theincreasing relative mass of the left ventricle, results in steepexponents across in-pouch development for the absolute volume(ml) of the myofibrils 1.38±0.10 (Fig. 3B), mitochondria 1.29±0.08(Fig. 3C), sarcoplasmic reticuli 1.45±0.13 (Fig. 3D) and T-tubules1.87±0.31 (Fig. 3E) (ANCOVA, F3,16=15.8, P<0.0001). Theincreasing prominence of these organelles across in-pouchdevelopment is in contrast to that of other cellular elements(primarily nuclei and cytosol), which decrease in volume-density(%) with an exponent of −0.44±0.12 (Table 1), such that absolutevolume (ml) increases with a relatively shallow exponent of 0.80±0.06 (Fig. 3F).During post-pouch development, the volume-density (%) of

cardiomyocytes within the left ventricle tissue does not vary, scalingwith an exponent of 0.00±0.04 (Table 1), and so absolute

cardiomyocyte volume (ml) scales in parallel with left ventriclemass, with an identical exponent of 0.78±0.15 (Fig. 3A). Thevolume-density (%) of key organelles within the cardiomyocytesalso scales invariantly with post-pouch body mass, with exponentsof 0.01±0.03 for myofibrils, −0.01±0.06 for mitochondria,0.01±0.09 for sarcoplasmic reticuli and 0.01±0.21 for T-tubules(Table 1) (ANCOVA, F3,32=0.026, P=0.99). Thus, the allometricexponents for the absolute volume (ml) of the myofibrils 0.79±0.15(Fig. 3B), mitochondria 0.77±0.17 (Fig. 3C), sarcoplasmic reticuli0.79±0.20 (Fig. 3D) and T-tubules 0.79±0.30 (Fig. 3E) are allstatistically indistinguishable from one another across post-pouchdevelopment (ANCOVA, F3,32=0.0082, P=1.00). The combinedvolume-density (%) of other cellular elements also scales with post-pouch body mass with an exponent not significantly different fromzero, −0.05±0.32 (Table 1), and the absolute volume (ml) scaleswith an exponent of 0.73±0.27 (Fig. 3F). Given that organellevolume-densities do not vary systematically with body mass inpost-pouch kangaroos, we can provide mean values, which are:

Oth

er c

ellu

lar v

olum

e (m

l)M

yofib

ril v

olum

e (m

l)

B1000

100

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Car

diom

yocy

te v

olum

e (m

l)

A1000

100

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Body mass (kg)

T-tu

bule

vol

ume

(ml)

E10

1

0.1

F100

10

1

0.1

1 10 1000.1

Mito

chon

dria

l vol

ume

(ml)

C100

10

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0.1

1 10 1000.1

Sar

copl

asm

ic re

ticul

um v

olum

e (m

l) D10

1

0.1

0.01

0.01

Vcm (post-pouch)�Mb0.78±0.15

Vcm (in-pouch)�Mb1.25±0.06 Vmf (in-pouch)�Mb

1.38±0.10

Vmf (post-pouch)�Mb0.79±0.15

Vmt (post-pouch)�Mb0.77±0.17

Vmt (in-pouch)�Mb1.29±0.08

Vsr (post-pouch)�Mb0.79±0.20

Vsr (in-pouch)�Mb1.45±0.13

Vtt (post-pouch)�Mb0.79±0.30

Vtt (in-pouch)�Mb1.87±0.31

Vother (post-pouch)�Mb0.73±0.27

Vother (in-pouch)�Mb0.80±0.06

Fig. 3. Scaling of cardiomyocyte and organelle volumes in the left ventricle of in-pouch and post-pouch M. fuliginosus. Open circles, in-pouch (N=6);filled circles, post-pouch (N=10). Regressions are plotted for the volume of (A) cardiomyocytes Vcm, (B) myofibrils Vmf, (C) mitochondria Vmt, (D) sarcoplasmicreticuli Vsr, (E) T-tubules Vtt and (F) the sum of other less abundant cellular elements Vother (see Materials and methods for list). See Table 1 for full statistics.

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63.7±1.3% for myofibrils, 22.7±0.8% for mitochondria, 2.9±0.1%for sarcoplasmic reticuli, 4.9±0.6% for T-tubules and 5.8±1.0% forthe combined sum of other less abundant cellular elements.

DISCUSSIONAll animals experience life history events during development thathave important consequences for physiology, activity and behaviour.Inevitably, such events also have important consequences foranatomy. For instance, birth in placental mammals necessitates ashift from placental to pulmonary gas exchange, which requiressignificant cardiovascular remodelling so that the left and rightventricles can transform fromworking in-parallel at similar pressures,to working in-series at vastly different pressures owing to differentresistances of the systemic and pulmonary circuits (Stopfkuchen,1987). This event is reflected in allometric studies of heart massgrowth, with placental mammals tending to show different scalingexponents before and after parturition (Hirokawa, 1972; Mitchell andSkinner, 2009).In marsupial mammals, cardiovascular remodelling also takes

place at birth, but this happens much earlier in development than itdoes in placental mammals (Runciman et al., 1995). While birth inmarsupials involves the energetic demands of crawling from thebirth canal to the pouch, it has been suggested that gas exchange ispredominantly via the skin at this stage (Mortola et al., 1999).Perhaps the most important cardiovascular life history event formarsupials occurs when the emergent young vacates the maternalpouch. In kangaroos and other marsupials, the period leading up topermanent pouch exit is associated with the development ofendothermy and an increase in metabolic rate (Hulbert, 1988; Roseet al., 1998). Around the same time, kangaroos make briefexcursions from the pouch, which continues until the animal iscapable of sustained and independent locomotion (Dawson, 2012).Reflecting this change in physiology, activity and behaviour, werecently showed that heart mass growth in the kangaroo shiftsallometric trajectory around the time of pouch exit (Snelling et al.,2015). A biphasic scaling pattern is further apparent in the subset ofanimals selected for the present study, where left ventricle massincreases with a relatively steep exponent of 1.18 during in-pouchdevelopment, before shifting to a relatively shallow exponent of0.78 across post-pouch development (Fig. 1).In addition to the biphasic scaling of left ventricle mass across

kangaroo development, it is apparent that cardiac growth is associatedwith significant changes at the cellular and subcellular levels(Figs 2, 3). In particular, in-pouch development is accompanied bysignificant changes to cardiomyocyte composition as the cells matureto a post-pouch-like state. The volume-density of myofibrils,mitochondria, sarcoplasmic reticuli and T-tubules within thecardiomyocytes increases significantly during in-pouch growth(Table 1), which is entirely consistent with the increasing volume-densities of these organelles in placental mammals during the fetaland early postnatal periods of development (Canale et al., 1986;Smolich, 1995). The volume-density and absolute volume of themuscle’s contractile machinery, the myofibrils, increases with steephyperallometry across in-pouch development (exponents of 0.13 and1.38, respectively), which suggests that the left ventricle’s contractileperformance, and capacity to generate pressure and blood flow, alsoincreases across this period. Consistent with this increase in myofibrilvolume-density during early development in kangaroos and placentalmammals, the tension (=stress; Nm−2) developed by isolated cardiacmuscle performing isometric (fixed muscle length) contraction isgreater in adult sheep comparedwith fetal lambswithin a range of pre-determined sarcomere lengths, over which the extent of actin–myosin

cross-bridging varies. In addition, maximum isometric tension,elicited at sarcomere lengths that maximise cross-bridging, isapproximately 60% greater in the adult compared with the fetalmyocardium (Friedman, 1972). Likewise, measurements taken fromisolated sheep myocardium performing isotonic (changing musclelength) contraction shows that both the extent and velocity ofshortening are greater in the adult than the fetal heart over a range ofapplied loads (Friedman, 1972). From awhole-organ perspective, theincreasing volume-density of myofibrils is consistent with theobservation that stroke volume is significantly depressed in neonatelambs at afterloads that are modest for adults (Downing et al., 1965).From a whole-animal perspective, the hyperallometry of myofibrilvolume-density and absolute volume, and the associated increase inrelative cardiac contractile performance, would augment cardiacoutput during in-pouch development and in the lead up to pouchvacation, and thus almost certainly facilitate the kangaroo’s transitionfrom ectothermy to endothermy (Hulbert, 1988; Loudon et al., 1985;Rose et al., 1998) and independent locomotion (Dawson, 2012).

During contraction–relaxation cycles in skeletal muscle,approximately 70% of the total energy harnessed from ATPhydrolysis is used by myofibril ATPase, while the remaining 30% isused by the sarcoplasmic reticulum for Ca2+ uptake and by thesarcolemma for the maintenance of ion gradients (Hoppeler andBilleter, 1991; Rall, 1985). Presumably, similar fractions apply tocardiac muscle. The bulk of this ATP energy is generated fromoxidative phosphorylation, which takes place within the cell’smitochondria. Thus, the increasing volume-density of myofibrilsduring in-pouch development will only translate to improvedcontractile performance provided sufficient mitochondria arepresent to supply the ATP. Thus, concomitant with the increasingmyofibril density, the volume-density and absolute volume ofmitochondria also increase with hyperallometry during in-pouchgrowth (exponents of 0.04 and 1.29, respectively). Nonetheless, theexponent for mitochondria is not quite as steep as that formyofibrils, such that the mitochondria:myofibril ratio decreasesacross in-pouch body mass, with an exponent of −0.09. This meansthat younger in-pouch individuals have more mitochondria relativeto myofibrils compared to older in-pouch individuals. It is feasiblethat in the younger in-pouch kangaroos, other energy-intensiveprocesses might be operating that demand mitochondrial ATP, inaddition to those involved in the contraction–relaxation cycle.During early development, particularly when muscle contractionsare weak and growth is rapid, an important role of mitochondriawould be to supply ATP energy for cardiomyocyte growth. Thedecreasing mitochondria:myofibril ratio during in-pouchdevelopment might also be explained by the apparent increase inmitochondrial cristae surface-density over this period; however, themyofibrils also exhibit structural maturation early in development,whereby the myofilaments become organised and aligned (Canaleet al., 1986). Measurements of myofilament anisotropy andmitochondrial cristae surface-density across development are anavenue for future research.

The increasing volume-density of myofibrils and mitochondriaacross in-pouch development is accompanied by an even steeperincrease in the volume-density and absolute volume of thesarcoplasmic reticulum (exponents of 0.20 and 1.45, respectively).The steep hyperallometry of the sarcoplasmic reticulum probablyarises in part to function alongside the increasing myofibrilcomponent, with which there is an intimate functional association,and in part to counter the increase in diffusion distance associatedwith increasing cardiomyocyte diameter with growth. The in-pouchperiod of kangaroo development is also associated with a very steep

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increase in the volume-density and absolute volume of the T-tubulesystem (exponents of 0.62 and 1.87, respectively). T-tubules areinvaginations of the sarcolemma that function to send actionpotentials deep within the cardiomyocyte. In placental mammals,T-tubules are absent in very early fetal cardiomyocytes, and onlybegin to form later in fetal and postnatal development (Canale et al.,1986; Smolich, 1995). There is a close association between theT-tubule system and the sarcoplasmic reticulum, reflecting theirfunctional association in the release of Ca2+ that then triggerscardiomyocyte contraction. Sarcolemma depolarisation activatesL-type channels that permit the passive movement of Ca2+ into thecardiomyocyte, which then triggers Ca2+ release from stores withinthe sarcoplasmic reticulum (Fabiato, 1989; Näbauer et al., 1989). Thecytosolic Ca2+ binds with troponin, which alters the tropomyosinarrangement, so that the myosin head and actin filament can bind, andmyofibrillar contraction can proceed.When cardiomyocytes are smalland immature, trans-sarcolemmal influx of Ca2+ alone appears largelysufficient to enable the relatively modest tensions (Nm−2) to develop.However, as cardiomyocytes mature, increasing cardiomyocyte sizeand diffusion distance, and greater developed tensions, necessitate anincreasing reliance on the T-tubule system to activate the increasinglynecessary intracellular Ca2+ stores of the sarcoplasmic reticulum(Anderson, 1996; Fisher, 1994). In support of this, experimentalevidence shows that decreased extracellular [Ca2+] has a moresignificant effect on the contractile function of neonatal rabbitcardiomyocytes compared with adults, while ryanodine-interferenceof sarcoplasmic reticuli reduces contraction amplitude in adults morethan it does in neonates (Chin et al., 1990). It therefore seems likelythat both the increase in myofibril volume-density and associatedcalcium requirements, as well as the increase in cardiomyocytediameter, contribute to the steep hyperallometry of the sarcoplasmicreticulum and T-tubule system across in-pouch development.The hyperallometry of myofibril, mitochondrial, sarcoplasmic

reticulum and T-tubule volume-densities during in-pouchdevelopment ensures that cardiac architecture is fully developedby the time of permanent pouch exit as the kangaroo must nowsupport its own locomotion. It is clear that ultrastructural maturityhas been attained in the emergent young, because the volume-density of these key organelles does not continue to increase beyondthe in-pouch stage, but rather scales invariantly with bodymass afterpouch exit (Table 1). This invariant scaling means that theirrespective absolute volumes scale in parallel with left ventriclemass, with a relatively shallow exponent of approximately 0.78.Hypoallometry of heart mass, and presumably left ventricle mass,across postnatal development in placental mammals is notuncommon; for instance, heart mass in the rat scales with anexponent between 0.74 and 0.82 (Stewart and German, 1999; vonBertalanffy and Pirozynski, 1952). Nonetheless, we show for thefirst time, at least in the post-pouch western grey kangaroo, that noultrastructural changes occur on the subcellular level that in any waycompensate for the shallow exponent. Thus, it appears that adecrease in relative cardiac requirements occurs as body massincreases in these free-roaming kangaroos. This could be related tothe fact that hopping locomotion is energetically more efficient asbody size increases, as larger macropods have a greater capacity tostore energy in the tendons, ligaments and muscles of the hindlimbsand tail between each hop (Baudinette et al., 1992; Bennett, 2000;Dawson and Taylor, 1973). While pentapedal locomotion isexpensive relative to the low speeds over which the gait isemployed, the total metabolic cost of hopping is neverthelesshigher, despite its apparent efficiency (Dawson and Taylor, 1973). Ithas been hypothesised that if the efficiency of hopping locomotion

increases as body mass increases ontogenetically, then it couldunload some of the work of the heart as body mass increases ingrowing post-pouch western grey kangaroos (Snelling et al., 2015).The shallow post-pouch exponent might also reflect generally lowermaximum activity levels in larger kangaroos due to old age, becausegrowth appears persistent across the lifetime of medium- and large-sized species of macropod (Jarman, 1983; Sadleir, 1965). Theshallow exponent might also be related to the decreasing surfacearea-to-body mass ratio associated with growth, which could reducethe cardiac output required for the maintenance of bodytemperature, especially when ambient temperatures drop wellbelow the thermal neutral zone.

The principle of Laplace has been used by Seymour and Blaylock(2000) to explain the allometric scaling of left ventricle mass andgeometry across 24 species of mammal. Their study showed thatpressure loading on the left ventricle increases in a systematicmanner with body mass (volume loading increases in directproportion), which appears to necessitate a graded increase inrelative wall thickness to ensure wall stress is conserved atapproximately 8–20 kPa. Applying the principle of Laplace to thepresent ontogenetic situation is made complicated because we donot know how pressure loading on the left ventricle changesthroughout development in a marsupial. Nonetheless, we do knowhow the volume-density of the myofibril contractile machinery inthe left ventricle tissue changes across development in the kangaroo(Table 1) and we know that the left ventricle of a mature post-pouchkangaroo (myofibril volume-density of tissue Vvmf,t=55%)generates a mean arterial pressure of 12.3 kPa (92 mmHg) underlight anaesthesia (Maxwell et al., 1964). Myocardial contractileforce is dependent on myofibril volume-density (Friedman, 1972),and so if the radius of curvature and wall thickness are held constant,and a linear relationship is assumed between mean arterialpressure (P; kPa) and myofibril volume-density of the ventricletissue (Vvmf,t; %), an adjusted pressure value can be calculated usingthe function P=0.221×Vvmf,t. The adjusted mean arterial pressure,together with left ventricle wall volume (Vw; ml) and lumen volume(Vl; ml), can then be used to calculate mean fibre stress across theleft ventricle wall (σf; kPa) according to the principle of Laplace,

Body mass (kg)

Fibr

e st

ress

(kP

a)

100

1 10 1000.1

10

σf (post-pouch)�Mb–0.01±0.12

σf (in-pouch)�Mb0.18±0.15

Fig. 4. Scaling of fibre stress across the left ventricle wall of in-pouch andpost-pouch M. fuliginosus. Open circles, in-pouch (N=6); filled circles, post-pouch (N=10). The principle of Laplace is applied to the left ventricle using aspherical model (Arts et al., 1991). Wall fibre stress (σf; kPa), averaged acrossthe cardiac cycle, is calculated according to σf=P×[1/3 ln(1+Vw/Vl)]

−1, whereVw and Vl are left ventricle wall and lumen volume (ml), respectively, and P ismean arterial pressure (kPa) adjusted to account for varying volume-density ofmyofibril contractile machinery in the left ventricle tissue (Vvmf,t; %) accordingto the equation P=0.221×Vvmf,t (see Discussion).

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using the spherical model presented by Arts and colleagues,σf=P×[1/3 ln(1+Vw/Vl)]

−1 (Arts et al., 1991). The results of thisanalysis are presented in Fig. 4. Because left ventricle wall andlumen volume scale more-or-less in parallel with one another acrossboth in-pouch and post-pouch stages of development, wall stressvaries as a function of mean arterial pressure and myofibril volume-density. Thus, wall stress increases significantly with body mass(Mb; kg) during in-pouch development, 13.0Mb

0.18±0.15, concomitantwith the increasing myofibril fraction of the myocardium. In contrast,wall stress scales independent of body mass across post-pouchdevelopment, 19.0Mb

−0.01±0.12, as the invariant scaling of myofibrilvolume-density keeps the calculated pressure loading constant.In summary, the growth and development of the left ventricle in the

western grey kangaroo is characterised by two distinct allometricpatterns that shift at approximately 5–6 kg body mass, whichcoincides with pouch vacation in this species. During in-pouchdevelopment, themass of the left ventricle and the absolute volume ofthe myofibrils, mitochondria, sarcoplasmic reticuli and T-tubules allincreasewith steep hyperallometry. Furthermore, calculations of wallstress show that it too increases significantly during this period ofdevelopment. These changes are associatedwith a significant increasein relative contractile performance, which probably facilitates thedevelopment of endothermy and independent locomotion inemergent young as they prepare to leave the pouch. Upon leavingthe pouch, cardiac ultrastructure has reached maturity, and thevolume-density of the key organelles together with wall stress scaleindependent of post-pouch body mass. Nonetheless, the size of theleft ventricle and the absolute volume of these organelles scale withrelatively shallow hypoallometry. This implies that post-pouchkangaroos have reduced cardiac requirements as body massincreases, which could be because the energy required for hoppingis independent of speed, and the capacity for energy storage duringhopping could increase as the kangaroo grows. This idea requiresformal testing. We therefore validate our hypothesis that the volume-density of key organelles within the left ventricle’s cardiomyocytesincreases with hyperallometry during in-pouch development, butscales independently of body mass across post-pouch development.However, despite the left ventricle’s wall-to-lumen volume ratioremaining constant throughout development, the increasingmyofibrilvolume-densityacross in-pouchgrowth is probablyassociatedwith anincreasing capacity to produce pressure, which leads to increasingstress across the left ventricle wall as the young develop in the pouch.

MATERIALS AND METHODSAnimal collectionWe joined a planned management cull of western grey kangaroosMacropusfuliginosus melanops (Desmarest 1817), 80 km south of Adelaide,Australia. A licensed marksman shot all animals according to Australianlegal requirements and code of practice (www.environment.gov.au).Approval to scavenge organ and tissue samples was provided by theUniversity of Adelaide Animal Ethics Committee (S-2011-223). Anallometric analysis of whole-heart and chamber masses in these animalshas been published (Snelling et al., 2015). In the present study, a subset ofthese animals was selected for a more detailed investigation into theallometry of left ventricle geometry and cardiomyocyte ultrastructure,comprising six ‘in-pouch’ kangaroos (pouched young) and 10 free-roaming‘post-pouch’ kangaroos (young-at-foot, juveniles and adults). The animalswere selected so that a comprehensive range of development and body masswas obtained.

Left ventricle mass and geometryEach carcass was weighed by spring balance (2.0, 5.0, 20 or 100 kgcapacity; Salter, Australia) before the thoracic cavity was opened, and the

whole heart removed, emptied of blood, and rinsed clean with an isotonicsaline solution (0.90% w/v NaCl). The left ventricle, defined as leftventricular free wall+interventricular septum (Fulton et al., 1952; Joyceet al., 2004; Keen, 1955), was separated from the remainder of the heart andweighed to either 0.1 mg (ventricles<30 g; AE163, Mettler, Greifensee,Switzerland) or 1.0 mg (>30 g; 1265 MP, Sartorius, Göttingen, Germany).The left ventricle was bisected along the mid-equator, and random linearmeasurements of transmural wall thickness (×4), and inner (×2) and outer(×2) diameters were taken with digital callipers to 0.01 mm. Left ventriclelumen volume (ml) was calculated as 4/3×π×Ri

3, where Ri is mean innerradius (cm).

Left ventricle ultrastructureUnbiased estimates of the relative composition of the left ventricle’sultrastructure were obtained by generating isotropic uniform randomimages for stereological analysis (Howard and Reed, 1998; Mühlfeldet al., 2010). Soon after the heart was extracted from the thoracic cavity,∼1 mm3 samples of cardiac muscle were excised from two randomlocations within the left ventricle wall. The samples were immediatelyimmersed in a chemical fixative solution of 3% glutaraldehyde and3% formaldehyde in 0.1 mol l−1 phosphate buffer (pH 7.4), and leftovernight at 6°C. The following day, each sample was given a series ofbuffer rinses (6×10 min) followed by secondary fixation in a 2% aqueoussolution of osmium tetroxide (4 h). Next, each sample was given a seriesof distilled water rinses (3×20 min) before progressive dehydration inethyl alcohol in 10% incremental steps starting from 50% to 80%(10 min each) followed by consecutive immersions in 90% ethanol(2×10 min), 100% ethanol (2×10 min) and finally pure propylene oxide(2×10 min). Samples were then incrementally infiltrated with embeddingresin (Durcupan, Fluka, Switzerland) at ratios of 3:1, 2:2 and 1:3(propylene oxide:resin), each for a duration of 2 h, and then leftovernight at room temperature in pure resin. Each sample was then placedin a random orientation in individual embedding moulds where they werecovered with pure embedding resin and left to polymerise in a 70°C ovenfor 48 h.

One randomly oriented 70 nm ultrathin section was cut at a randomdistance into each sample using 8 mm glass knives, a 2.4 mm diamondknife (Ultra 45°, Diatome, Switzerland) and an ultramicrotome (EM UC6,Leica Microsystems, Germany). Ultrathin sections were placed onto 3 mmcopper mesh grids, stained with uranyl acetate (15 min) and lead citrate(10 min), and viewed with a 120 kV transmission electron microscope(Tecnai G2, FEI, USA) coupled to an in-column CCD digital camera(Veleta, Olympus, Japan) running the bundled TIA imaging platform. Fromeach section, 10 low magnification images (×1100–2550) of random leftventricle tissue and 10 high magnification images (×7900–20,500) ofrandom cardiomyocyte ultrastructure were captured and digitally stored.Magnification was adjusted to account for differences in cardiomyocytesize across development. In total, 640 random images were taken from 32randomly oriented sections, cut from 32 random samples sourced from theleft ventricle of 16 kangaroos.

Each image was imported into a computer graphics program(CorelDRAW 15, Corel Corporation, Canada). Low magnification imageswere analysed with a randomly superimposed point grid test system, and thetotal number of points falling on cardiomyocytes and non-cardiomyocytes(blood vessels, pericytes, fibroblast cells and collagen) were counted. Totalleft ventricle cardiomyocyte volume (Vcm; ml) was calculated for eachkangaroo:

Vcm ¼ Vvcm;t �Mlv � r�1; ð1Þ

where Vvcm,t is the fraction of left ventricle tissue occupied bycardiomyocytes determined from point grid counts, Mlv is the leftventricle mass (g) and ρ is muscle density (1.06 g ml−1; Mendez andKeys, 1960; Vinnakota and Bassingthwaighte, 2004). High magnificationimages were also analysed with a randomly superimposed point grid testsystem, and the number of points falling on myofibrils, mitochondria,sarcoplasmic reticuli, T-tubules and other less abundant cellular elements(e.g. nuclei, cytosol, sarcolemma, glycogen, lipid) were counted. Their

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respective volumes in the left ventricle (Vorg; ml) were calculated as:

Vorg ¼ Vvorg;cm � Vcm; ð2Þwhere Vvorg,cm is the fraction of cardiomyocyte occupied by the organelle(or the sum of other cellular elements) determined from point grid countsand Vcm is the total left ventricle cardiomyocyte volume (ml) calculated inEqn 1.

Statistical analysesAll mean values are presented with 95% confidence intervals. Body mass isexpressed in kg, heart and left ventricle mass are in g, linear dimensions arein mm, volumes are in ml and volume-densities are expressed aspercentages. Allometric relationships were derived by taking the log10 ofthe variable and of the body mass, and calculating ordinary least-squareslinear regressions. The slopes and intercepts of the regressions werecompared with ANCOVA, with the morphometric variable of interest set asthe dependent, and body mass as the covariate (Zar, 1998), using statisticalsoftware (GraphPad Prism 6, GraphPad Software, USA). To determinewhether biphasic allometry existed in the dataset, a breakpoint analysis wasperformed by fitting a series of two-phase linear regressions to thelog10-transformed data in which the intersection point was shifted alongconsecutive readings. The breakpoint was identified as the intersection thatminimised the sum of the regressions’ residual sums of squares (Mueller andSeymour, 2011; Yeager and Ultsch, 1989).

AcknowledgementsThe authors acknowledge exceptional and humane marksmanship exhibited duringthe management cull and we thank the marksman for allowing us access tocarcasses.We thank Dr David McLelland from Zoos South Australia who engaged indiscussions on the scaling of heart size. We are indebted to volunteers who assistedin the field. We thank John Snelling from the University of Adelaide for donation of autility vehicle to collect carcasses. Two reviewers provided valuable feedback on themanuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsE.P.S. and D.A.T. are largely responsible for the project, working from a concept ofR.S.S., S.K.M. and A.P.F. Tissue fixation and electron microscopy were performedby E.P.S., C.M.L., L.W. and R.W. Data were analysed and interpreted by E.P.S.,D.A.T. and R.S.S. with additional input from all authors. All authors drafted andrevised the manuscript.

FundingThis research was supported by the Australian Research Council [DP-120102081 toR.S.S., S.K.M., and A.P.F.]. A.P.F. holds a Canada Research Chair.

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