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Articles in PresS. Am J Physiol Heart Circ Physiol (April 8, 2005). doi:10.1152/ajpheart.00866.2004
Structural differences in two biochemically-defined populations
of cardiac mitochondria
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Alessandro Riva , Bernard Tandler , Felice Loffredo , Edwin Vazquez , and Charles
Hoppel
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Department of Cytomorphology, School of Medicine, University of Cagliari, I-09042 Cagliari,
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Italy; Department of Biological Sciences, School of Dental Medicine, Case Western Reserve
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University, Cleveland, Ohio, 44106; Departments of Pharmacology and Medicine, School of
Medicine, Case Western Reserve University, and Louis Stokes Veterans Affairs Medical
Center, Cleveland, Ohio 44106
Address for reprint requests and other correspondence:Charles Hoppel, MD, Louis Stokes
VA Medical Center, Medical Research Service (151W), 10701 East Boulevard, Cleveland,
Ohio 44106 (E-mail: [email protected]).
Copyright © 2005 by the American Physiological Society.
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Alessandro Riva, Bernard Tandler, Felice Loffredo, Edwin Vazquez, and Charles
Hoppel. Structural differences in two biochemically-defined populations of cardiac
mitochondria. Am J Physiol Heart Circ Physiol xxx: xxx-xxx-To determine if there are
structural differences in two topologically separated, biochemically-defined mitochondrial
populations in rat heart myocytes, the interior of these organelles was examined by high
resolution scanning electron microscopy. Based on a count of 159 in situ subsarcolemmal
mitochondria (SSM) (those that directly abut the sarcolemma), these organelles possess
mainly lamelliform cristae (77%), whereas the cristae in in situ interfibrillar mitochondria (IFM)
(those situated between the myofibrils) (300 counted) are mainly tubular (55%) or a mixture
of tubular and lamelliform (24%). Isolated SSM (374 counted), like their in situ counterparts,
have predominantly lamelliform cristae (75%). The proportions of crista types in isolated IFM
(337 counted) have been altered, with only 20% of these organelles retaining exclusively
tubular cristae, whereas 58% are mixed-- of the latter, lamelliform cristae predominate. This
finding suggests that in contrast to SSM, the cristae in IFM are structurally plastic, changing
during isolation. These observations on more than a thousand organelles provide the first
quantitative morphological evidence for definitive differences between the two populations of
cardiac mitochondria.
high resolution scanning electron microscopy; cardiomyocytes; heart; cristae
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The mitochondria of cardiomyocytes consist of two spatially disparate populations: one
abutting the sarcolemma, the other trapped within the contractile apparatus. Techniques that
permit the separate isolation of these two populations have led to the finding that in several
mammalian species the two show significant physiological differences.6,7,10 Despite these
documented biochemical differences, studies based on conventional transmission electron
microscopy (TEM) have failed to reveal morphological disparities between the two
populations. We have used a recently developed osmium-extraction technique for scanning
electron microscopy that allows inspection at high resolution (HRSEM) of the interior of
cells.16 With this methodology, we found that cardiac mitochondria have different cristae that
characterize each population. These differences are maintained, albeit to a lesser degree, in
isolated mitochondria separately derived from the two sets of cardiac mitochondria. High
voltage electron microscope tomography (HVTEM)3,8,9,12-14 has paved the way for a
reexamination of cristal structure in mitochondria in other tissues, but HRSEM permits
quantitative estimation of crista morphology in a very large sample compared to the very
limited number of tomographic reconstructions.
Materials and Methods
Animals. Six Sprague -Dawley rats (Charles River Laboratories, Wilmington, MA) weighing
330-690 g (2.5 - 6 months of age) were used in this study. The rats were killed by
decapitation and the hearts extirpated. The protocol and animal care were reviewed and
approved by the Institutional Animal Care and Use Committees of the Louis Stokes VA
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Medical Center Medical Research Service and Case Western Reserve University School of
Medicine.
Mitochondrial Isolation and biochemistry. A portion of the left ventricle from each heart
was retained for microscopic examination. The remainder (left and right ventricle, but not
atria) of each heart from four of the six rats was used to isolate both subsarcolemmal
mitochondria (SSM) and interfibrillar mitochondria (IFM) according to Palmer et al.10 except
that Chappell-Perry buffer was used. Oxidative phosphorylation was studied as previously
described2,10.
Scanning Electron Microscopy. For HRSEM16, fresh, full-thickness strips measuring 3 x 5
mm, of left ventricular tissue were fixed for 15 minutes at room temperature in a mixture of
0.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1M cacodylate buffer. The tissue was
rinsed in three changes of PBS, each for 10 minutes. Specimens were postfixed for two
hours in the dark at 4oC in a 1:1 mixture of 2% osmium tetroxide and 1.25% potassium
ferrocyanide. After once again rinsing in three changes of PBS, the tissue was embedded in
1% agarose and cut with a TC2 Sorvall tissue sectioner into sections 150 µm thick. These
sections were rinsed three times in PBS and subjected to a second postfixation in
ferrocyanide-reduced osmium for one hour in the dark, rinsed three times in PBS. The
sections were macerated (osmium-extracted) for 44-48 hours in 0.1% OsO4 at 25oC, then
rinsed in PBS. They were dehydrated in ascending concentrations of acetone, then critical
point-dried using carbon dioxide. Mitochondrial pellets were processed in almost the same
fashion as were solid tissues except that the former were embedded in agarose and fractured
with liquid nitrogen. The sections or pellet fragments were coated with platinum in an Emitech
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575 sputtering apparatus and examined in an FE Hitachi S4000 scanning electron
microscope operating at 15-20 kV.
Quantitation. To classify and quantitate in situ mitochondrial types according to their cristal
structure, only transversely-sectioned cardiomyofibers with intact sarcolemmas were
included. Mitochondria were considered as subsarcolemmal only if they abutted the
sarcolemma. Interfibrillar mitochondria were those some distance removed from the
sarcolemma. Two observers independently counted those mitochondria whose intracellular
location was clear-cut according to the foregoing criteria. In the case of the mitochondrial
pellets, mitochondria were counted in micrographs of random fields.
Statistical Analysis. Results of oxidative phosphorylation were expressed as mean ±
standard deviation (SD). Differences between the IFM and SSM were compared using
Student's t-Test. For the Scanning EM data a 2X2 Chi-square test was performed comparing
lamellar versus tubular cristae in SSM and IFM both in situ and in isolated mitochondria. A p
value < than 0.05 was considered significant.
Results
Figure 1A shows a cardiac muscle fiber in transverse section; the transected
sarcolemma is retained. The contractile apparatus has been extracted in its entirety; the gaps
between columns of mitochondria represent the space originally occupied by myofibrils.
Those mitochondria immediately subjacent to and abutting the sarcolemma correspond to
subsarcolemmal mitochondria (SSM) (Fig. 1B) based on TEM. In contrast, those
mitochondria situated amid the gaps correspond to the interfibrillar mitochondria of TEM
(Fig.1C). To quantitate types and subcellular location of in situ mitochondria, scoring was
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carried out only on tranversely-sectioned cardiomyofibers by two investigators counting
independently. In order to identify SSM versus IFM, only those fibers where the sarcolemma
was clearly visible (as in Fig. 1A) were examined. Further, only those mitochondria whose
intracellular position was unambiguous were counted.
Based on examination of multiple sections of four hearts, 77% of the SSM had
lamelliform cristae (Fig 2A,B; 3A). Lamelliform cristae are broad and flat, may show throughand-through fenestrations, and are joined to the boundary membrane by numerous crista
junctions, to use the terminology of Perkins et al.14 Some of the lamelliform cristae have small
vesicular protuberances along their free edges; these may represent transected crista
junctions (Fig. 2b).
Crista structure in the in situ IFM differs from that in SSM. On the one hand, some
mitochondria possess only tubular cristae (Figs. 2C,3A), whereas the remainder contain one
or several lamelliform cristae interspersed among the tubular ones (Fig. 2D), or may have
only lamelliform cristae (Fig. 3A). Such flat cristae display an abundance of crista junctions.
Crista junctions as such are not identifiable in the tubular cristae, but this may be due to the
fact that the overall diameter of these cristae is a constant so that even if crista junctions are
present they do not appear as a constricted portion of these inner membrane structures. The
tubular cristae frequently branch and occasionally anastomose with one another, sometimes
forming a lattice (Fig. 2D). Fifty-five percent of the IFM have tubular cristae exclusively,
although another 24% have tubular cristae among which are lamelliform cristae (Fig.3A).
Some of the tubular cristae are fingerlike whereas others are disposed in lattices. Twenty-one
percent of the IFM had only lamelliform cristae.
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In summary, our observations show that although most cristae in in situ SSM are
lamelliform, an occasional tubular crista occurs in these organelles. In contrast, the polar
opposite of this observation occurs in IFM where flat cristae occur among the predominant
tubular ones (Fig. 3A).
Our metabolic studies of isolated cardiac mitochondria paralleled our previous
published work (10). These data are shown in Table I. The yield of mitochondrial protein in
the SSM and IFM, as well as their state 3 and 4 rates of oxidation (data not shown),
respiratory control ratios, ADP/O ratios, and maximal ADP-stimulated rates of oxidation are
virtually identical to those values reported earlier in adult rats (2,10).
To quantitate the morphology of isolated mitochondria of each population, micrographs
of random fields were scored in terms of cristal organization in the same fashion as the in situ
organelles.The structure of isolated SSM is nearly identical to that of in situ SSM, with most
(75%) having lamelliform cristae (Figs. 3B, 4A). Of the balance of the isolated SSM, most had
a mix of lamelliform and tubular cristae (Fig. 4B). Compared to the in situ situation, the
percentage of IFM with tubular cristae (Fig. 4C) has been halved (20%) , whereas the
percentage of mitochondria with heterogeneous cristae (Fig. 4D) has doubled (58%) (Figs.
3A,B). Virtually the same percentage of IFM with lamelliform cristae as that prevailing in situ
is retained in the isolated organelles (Figs. 3A,B)
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Discussion
High resolution scanning electron microscopy combined with improved osmium
extraction techniques have led to the uncovering of certain structural features of rat heart
mitochondria not previously reported by TEM. Subsarcolemmal mitochondria have
predominantly lamelliform cristae, whereas interfibrillar organelles have cristae that mainly
are tubular or that have a mixture of tubular and lamellar ones. Our study indicates that
isolated SSM retain, to a large extent, virtually identical cristae, i.e., lamelliform, that they
exhibit in situ. In contrast, there is in isolated IFM a doubling in the number of those
organelles that have heterogeneous cristae and a reciprocal decrease in the number of
mitochondria with only tubular cristae. One interpretation of these observations is that cardiac
mitochondrial cristae are interconvertible in terms of structure. If this is true, then what is the
signal for conformational change? Are changes in crista structure and mitochondrial function
in lock step? Model systems exist that could be used to address this latter issue. In hearts of
cardiomyopathic hamsters6 and of aged rats2 IFM exhibit a selective decrease in oxidative
phosphorylation, but TEM has failed to detect ultrastructural alterations in this mitochondrial
population; HRSEM examination of cristae in osmium-extracted IFM in aged hearts currently
is under investigation.
In brown fat mitochondria, which previously have been examined by means of high
voltage transmission electron microscopy (HVTEM) tomography,8 our results obtained with
HRSEM16 are identical to those obtained by the former method. In such mitochondria, we
have noted the same arrangement of cristae and crista junctions with the same resolution as
in HVTEM tomography. A major advantage of our methodology is that numerous organelles
(literally, hundreds) can be observed in a single specimen without the laborious
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reconstructions (that limit sample size) required for HVTEM tomography. Because osmiumextraction is carried out on thoroughly-fixed sections rather than on whole specimens, there is
no concentration gradient of osmium set up during extraction-- rather, all areas of a given cell
are simultaneously exposed to this agent, thus eliminating the distance from the extracellular
space as a factor in the morphological differences that we report here. We and others using
HRSEM examined the ultrastructure in other tissues of an assortment of membranous
organelles including rough endoplasmic reticulum,15,16,18 Golgi apparatus,16,18 annulate
lamellae,16 and secretory granule,15,16 and, in every case, found these organelles to precisely
match their TEM counterparts. Moreover, we have used our method to observe mitochondria
in a very large number of organs and have concluded that osmium extraction is no more
likely to produce artefacts than is any other ultrastructural technique currently in use. So, in a
sense, HRSEM and HVTEM tomography are not only compatible, but complementary as
well.
The morphological differences we describe between the two populations of cardiac
mitochondria from normal hearts parallel the biochemical differences in these two sets of
organelles. The rate of oxidative phosphorylation in the IFM is about 150% the rate observed
in the SSM10. Both populations of isolated mitochondria are well-coupled and have excellent
ADP/O ratios, marking these mitochondria as functionally normal. An additional biochemical
difference is that the specific activity for many, but not all enzymes, also is 150% greater in
the IFM than in the SSM.10 How do these biochemical differences relate to the morphological
observations? Although IFM have higher specific activities of some enzymes, these enzymes
are not controlling for oxidative phosphorylation and thus the basis for the enhanced activity
of the IFM must lie elsewhere. What advantages are conferred upon the IFM by having
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tubular rather than flat cristae? One possibility is that reduction of the intracristal space of
tubular cristae leads to a higher concentration of protons within these structures, thus
enhancing ATP synthase activity, which facilitates oxidative phosphorylation. A case in point
is the alteration in mitoplast morphology occasioned by changing oxidative state12. An
additional possibility is that key molecular interactions within the electron transport chain
complexes situated in crista membranes are affected by membrane conformation. For
example, Schàgger17 has described ‘supercomplexes,’ assemblies of the electron-transport
chain complexes that require close association to operate at maximum efficiency. The
possibility also exists that the biochemical composition of the two types of cristae differs to
some extent. The phospholipid or protein composition of their membranes might play an
important role in determining crista morphology. Dynamin1,4 and ATP synthase11 have been
shown to influence cristae morphology in yeast and in several mammalian tissue culture cell
types. The current emphasis on mitochondrial proteomics and lipidomics could lead to data
relevant to this point.
ACKNOWLEDGMENTS
We thank Drs. Giacomo Diaz and Alan Davis for statistical assistance and Drs. Edward
Lesnefsky, John Mieyal, and Medhat Hassan for reviewing the manuscript. Gabriele Conti,
Michela Isola, Francesco Loy, Marco Piludu, and Rachel Floyd provided technical assistance.
This work was supported in part by grants from a FIRB program and from the Italian Ministry
of Health, and by NIA grant P01 AG15585 and by the Department of Veterans Affairs Medical
Research Service.
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Perkins G, Renken C, Martone ME, Young SJ, Ellisman M and Frey T. Electron
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FIGURE LEGENDS:
Fig. 1. (A) A tranversely-sectioned osmium-extracted cardiomyocyte; its sarcolemma is
indicated by a series of arrows. Immediately within the sarcolemma are the SSM; the more
central organelles are the IFM. The empty spaces between the latter are sites formerly
occupied by myofibrils, which have been completely extracted. Scale line = 4 ųm. (B) A
portion of the sarcolemma from the preceding micrograph at higher magnification. The
serried mitochondria that abut the sarcolemma (arrows) are SSM. Scale line = 1 ųm. (c) IFM
from the box in Fig 1A at higher magnification. Scale line = 1 ųm.
Fig. 2. Osmium-extracted in situ mitochondria, as seen by HRSEM. (A) An SSM with
lamelliform cristae. (B) An SSM showing an en face view of a lamelliform crista, which is
fenestrated. (C) An IFM with digitiform cristae. (D) From this vantage point, the tubular cristae
in this IFM are seen to form a lattice. Scale lines = 0.5 ųm.
Fig. 3. Quantitation of mitochondrial types based on cristal morphology. (A) SSM: clear bar, in
situ; solid bar, isolated SSM. (B) IFM: clear bar; in situ; solid bar, isolated IFM. Statistical
analysis was done using a 2X2 Chi-square test. *Lamellar versus tubular cristae in in situ
and in isolated mticohondria p<0.0001 for SSM and IFM. †Comparing cristae in in situ
versus isolated mitochondria (both SSM and IFM) p is > 0.1 and not significant.
Fig. 4. Osmium-extracted isolated mitochondria. The strands in the background are ribbons
of agarose. (A) A cluster of SSM with solely lamelliform cristae. Scale line = 1 ųm. (B). An
SSM with mostly lamelliform cristae. Scale line = 0.5 ųm. (C) An IFM with exclusively tubular
cristae. Scale line = 0.5 ųm. (D) This IFM has mainly lamelliform cristae, but also has a
cluster of fingerlike ones, seen here in cross-section. Scale line = 0.5 ųm.
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Table I
Protein yield (mg/g wet wt) and Oxidative Metabolism in SSM and IFM (nAO/min/mg)
Yield (mg/g)
SSM
12.9 ± 3.1
IFM
13.8 ± 2.7NS
RCR
5.9± 1.4
8.5 ± 2.3NS
ADP/O
3.18 ± 0.26
3.20 ± 0.93NS
ADP
262 ± 45
463 ± 87*
DNP
270 ± 48
488 ± 118*
Means ± SD. 20 mM Glutamate was used as the substrate.
ADP, oxidative phosphorylation with 2 mM ADP.
DNP, uncoupled respiration stimulated by dinitrophenol.
NS
not significant vs SSM
* p< 0.05 vs SSM
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Figure 1
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Figure 2
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Figure 3
A
percentage
100
subsarcolemmal
In Situ
75
Isolated
50
25
0
lamelliform
B
percentage
100
mixed
tubular
mixed
tubular
interfibrillar
75
50
25
0
lamelliform
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Figure 4
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