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High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43.

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THE ANATOMICAL RECORD PART A 288A:1059–1067 (2006)
High-Pressure Freezing and Freeze
Substitution of Rat Myocardium for
Immunogold Labeling of Connexin 43
CHRISTIAN MÜHLFELD* AND JOACHIM RICHTER
Division of Electron Microscopy, Department of Anatomy,
University of Göttingen, Göttingen, Germany
ABSTRACT
The value of high-pressure freezing (HPF) and freeze substitution
(FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14day-old rats at baseline and at 15, 30, 45, and 60 min after induction of
ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for
detection of Cx43. Gold particles associated with gap junction areas, free
plasma membrane, and annular gap junctions (AGJs) were counted and
distributions compared by contingency table analysis. HPF and FS
resulted in excellent preservation of antigenicity for Cx43. The mostly good
preservation of the ultrastructure was limited by mechanical damage at
the border and by ice crystal formation in the center of the tissue blocks.
In normal myocardium of newborns, gold particles associated with free
plasma membrane were frequently observed, with AGJs only seldom. In
older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats,
however, ischemia induced a shift of Cx43 from gap junction plaques to
AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the
process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated
by ischemia in an early developmental state. Anat Rec Part A, 288A:
1059–1067, 2006. Ó 2006 Wiley-Liss, Inc.
Key words: high-pressure freezing;
stereology; connexin 43
High-pressure freezing (HPF) has been introduced into
transmission electron microscopy (EM) as a method of
rapidly fixing small specimens without prior chemical fixation (McDonald, 1999). In comparison with aldehyde fixation (routinely used in EM), HPF leaves the antigenicity
of the frozen specimens unaltered by the fixation process
and therefore seems to be an ideal tool for immuno-EM
(Monaghan et al., 1998). Further, it was shown that HPF
is superior to freezing at ambient pressure, which results
in formation of large ice crystals and consequently in destruction of tissue ultrastructure (Monaghan et al., 1998).
If high-pressure frozen tissue is further processed by
freeze substitution (FS) and embedded in methacrylates,
it should yield materials highly suitable for immuno-EM.
However, the specific value of HPF and FS for immunoÓ 2006 WILEY-LISS, INC.
myocardium;
ischemia;
EM may vary with the biological material of interest
(Nicolas and Bassot, 1993). The mammalian heart, for
instance, is mainly composed of cardiomyocytes that are
connected with several surrounding cardiomyocytes by
gap junctions. Investigations comparing immersion and
*Correspondence to: Christian Mühlfeld, Department of Anatomy, Division of Electron Microscopy, Kreuzbergring 36, D-37075
Göttingen, Germany. Fax: 49-551-397004.
E-mail: cmuehlf@gwdg.de
Received 12 May 2006; Accepted 17 July 2006
DOI 10.1002/ar.a.20380
Published online 1 September 2006 in Wiley InterScience (www.
interscience.wiley.com).
1060
MÜHLFELD AND RICHTER
perfusion fixation have shown that the mechanical damage by cutting out a piece of myocardium may lead to
alterations within the whole specimen (Gavin et al., 1991).
Since specimens suitable for HPF have to be of a very
small size (approximately 0.2 mm in diameter), this problem may arise here, too, although the time window from
biopsy to fixation can be reduced to less than 40 sec
(Hohenberg et al., 1996). To our knowledge, there is no
study that uses HPF and FS to investigate the distribution of a particular antigen in mammalian myocardium
systematically.
Therefore, we performed an immuno-EM investigation
on normal and ischemic myocardium taken from postnatal rats of different age groups using HPF and FS.
Immunogold labeling and ischemic damage of myocardium were evaluated by design-based stereological methods. The target antigen chosen for the present study was
the major myocardial gap junction protein connexin 43
(Cx43) because immunogold labeling of Cx43 is known to
be difficult in myocardium treated with crosslinking fixatives. Specifically, glutaraldehyde, even in low concentrations, markedly decreases Cx43 immunoreactivity (Luke
et al., 1989). Cx43 is synthesized at the rough endoplasmic reticulum and transported to the free lateral membrane of cardiomyocytes via the Golgi apparatus, from
where it is translocated to the specialized areas of the
gap junction plaques. Due to their high turnover, gap
junctions are frequently removed from the specialized
membrane areas and degraded by lysosomes or proteasomes. One classical feature of this degradation pathway
is the formation of annular gap junctions (AGJs), which
consist of a ring of double membranes where connexins
can be localized by immuno-EM (Dermietzel et al., 1991;
Saffitz et al., 2000; Segretain and Falk, 2004). We quantified gold particles associated with gap junctional and nonjunctional plasma membrane and AGJ to study whether
the distributions undergo changes during postnatal development and in the course of ischemia. This approach was
used for two reasons. First, it is well known that profound
remodeling processes of Cx43 distribution take place during postnatal development (Fromaget et al., 1992; Gourdie
et al., 1992; Peters et al., 1994). Starting with discontinuous gap junction plaques around the entire cell surface in
the newborn rat, after the first postnatal weeks the typical
localization of Cx43 at the longitudinal parts of the intercalated disks is reached. However, most studies on this
topic rely on light microsopy and no data exist concerning
possible changes in the preferential localization of Cx43 in
free sarcolemma, AGJs, and gap junctions, respectively.
Second, the redistribution of Cx43 during ischemia has
important clinical impact because of ischemia-associated
arrhythmias. Importantly, Cx43 has been shown to be an
essential part in the protective response of the myocardium to ischemic preconditioning (Schwanke et al., 2002).
In a previous study, Vetterlein et al. (2006) have shown
that ischemic preconditioning results in an increased
Cx43 immunoreactivity in the free plasma membrane of
cardiomyocytes. As neonatal hearts are more tolerant to
ischemia than those of adult rats (Singer, 1999) and neonatal ischemia tolerance shares similarities with ischemic
preconditioning, we hypothesized that the myocardium of
newborn rats might be equipped with a similar distribution like that shown by Vetterlein et al. (2006) for ischemic
preconditioning and that this distribution may get lost
during postnatal development.
TABLE 1. Number of animals, body and heart weight
0d
5d
14d
Number of
6
5
6
animals
Body weight (g)
6.16 6 0.18 16.59 6 1.07 33.08 6 2.37
Heart weight (mg)
42 6 4
124 6 9
209 6 18
Our study clearly demonstrates that HPF and FS yield
a good ultrastructural preservation combined with an
excellent preservation of antigenicity of Cx43. HPF and
FS may therefore represent an ideal alternative to aldehyde perfusion fixation of the heart, especially if the antigenicity of the structure of interest is vulnerable to aldehyde fixation. In accordance with our hypothesis, Cx43
immunoreactivity within the free plasma membrane was
markedly higher in newborn than in older rat hearts,
whereas the association of immunogold particles with
AGJs was increased in the older rat hearts. Ischemia did
only alter Cx43 distribution in the 5-day-old rats, a result
we cannot explain so far.
MATERIALS AND METHODS
Animals
The experiments described in this study comply with
the current German laws and conform to the Guide
for the Care and Use of Laboratory Animals published by
the U.S. National Institutes of Health (NIH publications
85-23, revised 1996). The painless sacrifice of the animals
was reported to the Bioethical Committee of the District
of Braunschweig, Germany.
Experimental Procedure
Postnatal rats of different ages (day 0, n ¼ 6; day 5, n ¼
5; day 14, n ¼ 6) were used in the present study. For further animal data, see Table 1. Animals were weighed, then
sacrificed either by decapitation (newborn) or by cervical
dislocation (5- and 14-day-old rats). The thorax was
opened by a median sternotomy, the rib cage was spread
by means of two needles and the beating heart was carefully excised, weighed, and washed in a Petri dish filled
with isotonic NaCl solution. The heart was then transferred to a new Petri dish filled with fresh NaCl solution
at room temperature and subjected to extracorporeal
global ischemia (Mühlfeld et al., 2006). Immediately after
this procedure, a biopsy was performed, taken from the
left ventricular free wall using the microbiopsy transfer
system described by Vanhecke et al. (2003) (Leica, Vienna,
Austria). After transfer of the specimen to a flat specimen
carrier, the latter was placed into a pod and inserted into
the cryofixation machine using a loading device. The specimen was then high-pressure-frozen at a mean pressure of
2,023 6 5 bar as described by Studer et al. (2001) (Leica
EM Pact). Further biopsies were performed after 15, 30,
45, and 60 min of ischemia, which underwent the same
procedure. In all cases, the time from biopsy to freezing
was approximately 30 sec.
For the comparison of HPF with freezing at manual
pressure and with chemical fixation, respectively, two
14-day-old rat hearts were used. For each fixation method,
three tissue blocks per animal were fixed. Freezing under
HIGH-PRESSURE FREEZING OF MYOCARDIUM
manual pressure was performed with the help of a forceps
precooled in liquid nitrogen. For chemical fixation, a fixative with 4% paraformaldehyde in 0.2 M HEPES buffer
was used.
Tissue Processing and Immunolabeling
The high-pressure frozen biopsy material was transferred to an AFS cryosubstitution unit (Leica, Bensheim,
Germany) precooled to 908C. They were then immersed
in 0.5% uranyl acetate in methanol for 72 hr, warmed to
458C at a rate of 58C/hr, and washed with methanol for
several times. Thereafter, specimens were removed from
the specimen carrier and immersed in Lowicryl HM20/
methanol 1:1 and 2:1 for 2 hr each. Finally, the specimens
were placed into Lowicryl HM20, which was polymerized
under UV light for 2 days at 458C. From these blocks,
ultrathin sections were cut and mounted either on onehole copper grids or on 100 mm nickel mesh grids. The
copper grid sections were stained with lead citrate and
uranyl acetate and used for stereological estimation of ischemic injury.
Ultrathin sections mounted on mesh grids were used
for immunogold labeling for Cx43. First, the grids were
floated on 0.02% glycine in Tris-buffered saline (pH 7.6)
for 15 min and then on blocking buffer containing 5% fetal calf serum, 0.1% Tween 20, 0.25% bovine serum albumin in Tris-buffered saline (pH 7.5) for 30 min. Afterward, the grids were transferred to the anticonnexin43
antiserum (diluted 1:50 in blocking buffer to a final
concentration of 20 mg/mL; catalog number MAB3068;
Chemicon, Canada) for 30 min. Grids were rinsed six
times each for 5 min with blocking buffer, and visualization of immunoreactivity was performed by incubation
with a secondary 10 nm gold-coupled antimouse antibody diluted 1:20 in blocking buffer (catalog number
EM.GAM10; British Biocell, U.K.). Grids were washed
four times for 4 min in distilled water, stained on a drop
of uranyl acetate, rinsed three times with distilled water,
and dried for 4 hr at 408C.
Control sections of normal and ischemic myocardium
were prepared by the same procedure as described above
except for the omission of the primary antibody. Apart
from very few gold particles without structural preference, all control sections were negative, i.e., no immunoreactivity of the secondary antibody with cardiomyocytes
was observed.
The chemically fixed probes were first immersed for
24 hr in 2.3 M sucrose and plunge-frozen before transfer
to the cryosubstitution unit. The protocol then applied to
the myocardium frozen under manual pressure or fixed
by aldehyde immersion was the same as the one applied
to the high-pressure frozen tissues.
Stereology and Electron Microscopy
All electron microscopic investigations were performed
with an EM 900 (Zeiss, Oberkochen, Germany) equipped
with a digital camera (Megaview III, Soft Imaging
Systems, Münster, Germany) and computer imaging
software (AnalySIS, Soft Imaging Systems, Münster,
Germany).
For each animal and time point, one section was investigated to evaluate parameters of ischemic damage. At a
primary magnification level of 7,0003, 60 images were
1061
obtained by systematic random sampling from each section. As ischemia is accompanied by swelling of myocyte
mitochondria and cardiomyocytes as a whole, the degree
of these alterations was established by an estimation of
the volume-weighted mean volume of mitochondria and
the cellular edema index as previously described in detail
(Mühlfeld et al., 2006). In short, a test system with 18
lines and 36 points was projected onto each test field.
Whenever a test point hit a mitochondrial profile, the
edge-to-edge chord length was measured along the corresponding test line. From these measurements, the volume-weighted mean volume of mitochondria was calculated according to Gundersen and Jensen (1985). Additionally, points hitting myofibrils, nuclei, mitochondria,
and sarcoplasm were counted to estimate the volume
densities of these compartments according to Weibel
(1979). From these volume densities, the cellular edema
index was estimated according to DiBona and Powell
(1980). This parameter increases when cardiomyocyte
swelling occurs.
Immunoelectron microscopy for Cx43 was employed to
estimate the mean number of gold particles associated
with one of the following compartments. The myocyte cell
surface was divided into specialized membrane area
where plasma membranes of two adjacent myocytes were
close enough to be bridged by two connexons, and nonspecialized membrane area where no direct contact with
other myocytes was present or where adjacent myocyte
membranes were divided by a gap too large to be bridged
by connexons. The third compartment under investigation consisted of AGJs, which were defined as intracytoplasmic organelles surrounded by two membranes. Only
once was an extracellular AGJ found and was not
included in the analysis.
For quantification of gold particles, one section per animal and time point was investigated. At a primary magnification of 7,0003, each section was scanned exhaustively by systematic random sampling. Thus, a total of
roughly 600 test fields (with an individual area of ca.
9 mm2) per section was used to count gold particles associated with one of the three compartments described above.
On each section, a total of approximately 150 gold particles was counted. The observed gold counts were used
to calculate mean gold counts for each time point and age
group. These mean gold counts were entered into the statistical analysis described below.
Statistics
To minimize the influence of the interindividual and
developmental composition of cardiomyocytes on the evaluation of ischemic swelling of mitochondria and cardiomyocytes, cellular edema index (CEI) and volumeweighted mitochondrial volume were normalized by
dividing these parameters of each time point by the normal value of the same animal, e.g., CEI (45 min)normalized ¼
CEI (45 min)/CEI (0 min). In order to analyze whether ischemia had induced a significant cell and mitochondrial
swelling over 60 min, Friedmans ANOVA was used. In
order to recognize those points in time that significantly
contribute to the swelling, the normalized CEI and mitochondrial volume were compared by the Wilcoxon test for
paired samples. In order to establish whether the degree
of ischemic damage differs at a given time point of ischemia among the age groups, the corresponding values of a
1062
MÜHLFELD AND RICHTER
TABLE 2. Contingency table analysis of gold counts in normal myocardium among the age groups
SpM
NSpM
AGJ
Column total
Nobs (Nexp)
0d
Nobs (Nexp)
5d
Nobs (Nexp)
14d
Row
total
partial v2
0d
partial v2
5d
partial v2
14d
87.2 (83.6)
19.2 (13.0)
3.3 (13.1)
109.7 (109.7)
67.8 (71.8)
13.4 (11.1)
13.0 (11.3)
94.2 (94.2)
137.0 (136.6)
12.7 (21.2)
29.5 (21.4)
179.2 (179.2)
292.0
45.3
45.8
383.1
0.153
2.984
7.307
10.444
0.223
0.465
0.265
0.953
0.001
3.407
3.032
6.441
In the first column, the specialised plasma membrane (SpM), non-specialised membrane (NSpM) and annular gap junctions (AGJs) compartments are denoted. For each of the compartments, a mean number of associated gold particles
was observed (Nobs). According to the equation given in the methods section the number of expected gold particles was
calculated (Nexp). To see whether the distributions of observed gold particles within the three compartments were
altered during postnatal development, partial v2 values and total v2 were calculated. For 4 degrees of freedom (3-1
compartments 3 3-1 groups) and with a total chi-squared value of 17.838 the null hypothesis that distributions are
similar has to be rejected. Those compartments that make a substantial contribution to total chi-squared (more than
10% of total), i.e. the intergroup difference are highlighted in bold letters within the table. Thus, gold particles with
non-specialised membrane are higher than expected in newborn rats and lower than expected in 14-day-old rats. The
opposite holds true for AGJ.
given point in time were compared by the Kruskal-Wallis
ANOVA.
The statistical evaluation of the immunoelectron microscopic data was performed according to Mayhew et al.
(2004). The observed numbers of gold particles were compared by use of a contingency table analysis to depict distributional changes within one age group during the progression of ischemia or among the age groups in normal
myocardium. From the number of observed particles, the
number of expected particles was calculated according to
the equation: column total 3 row total/grand total ¼
expected number of gold particles. Partial chi-square values for each group and compartment are given by: (observed golds expected golds)2/expected golds. The total
chi-square value makes it possible to determine whether
the observed distributions differ significantly. If the observed gold distributions are different, the partial chisquare values will indicate those compartments that make
a substantial contribution to the total chi-square value,
i.e., the difference between groups. A substantial contribution is given by 10% or more of the total chi-square value
(Mayhew et al., 2004). An example for this evaluation is
reported for the age group differences of Cx43 distribution
(Table 2).
RESULTS
Preservation of Ultrastructure and Antigenicity
The preservation of myocardial ultrastructure by the
given protocol was heterogeneous, as can be seen in
Figures 1, 4, and 5. The quality of preservation depended
on the age and the depth of the tissue block from which
the section was cut. Mechanical damage at the border of
the tissue block affected larger tissue volumes in the
older rats due to the larger cell size. Ice crystal formation
was frequently observed in the center of the tissue block
commencing at a depth of approximately 75 mm. Ice crystal formation began nearer to the surface in tissue blocks
from newborn than from older rats. Therefore, this artifact was more frequently observed in newborns than in
older rats.
In several sections obtained between 20 and 75 mm, a
good ultrastructural preservation was found where neither damage due to mechanical distortion nor ice crystal
formation was seen (Fig. 1a). However, in several sections,
Fig. 1. Immunogold labeling of gap junctions. a: 60 min of ischemia.
In 14-day-old rats, gap junction plaques (thin arrows) were usually in
close connection with adherens junctions (white asterisks). Note the division of membranes after the gap junction area (thick arrow). b: 15 min
of ischemia. In newborn rats, no conncetion with adherens junctions
was observed. Gap junctions between adjacent myocytes (thin arrows)
were interrupted by areas of nonjunctional membrane (thick arrows).
HIGH-PRESSURE FREEZING OF MYOCARDIUM
1063
Fig. 3. Antigenicity in chemically fixed myocardium. The myocardium shown here was fixed with 4% paraformadehyde in 0.2 M HEPES
buffer and shows Cx43 immunogold labeling of the longitudinal part
(arrows) of an intercalated disk. The white asterisks mark the transversal parts of the intercalated disk. Although specific, the labeling was
rather weak compared with HPF (see Fig. 1a for comparison). mi, mitochondria; mf, myofibrils.
Immunogold Labeling
Fig. 2. Ice crystals in high-pressure frozen and manually frozen
myocardium. a: Normal myocardium, 14-day-old rat. High-pressure frozen myocardium was usually well-preserved. However, within some
areas, ice crystals (arrows) were observed that mainly affected the
ultrastructure of myofibrils (mf) and nuclei (nuc). Mitochondria (mi) were
usually not altered. b: Normal myocardium, 14-day-old rat. Freezing
under manual pressure led to a disturbed ultrastructure with large ice
crystal formation. Nuclei (nuc) and myofibrils (mf) were completely distorted, whereas mitochondria (mi) and the plasma membrane (arrows)
were damaged less severely.
ice crystal formation was present in varying degrees
(Fig. 2a). In comparison to myocardium fixed by manual
freezing followed by freeze substitution (Fig. 2b), however,
high-pressure freezing always yielded better results.
The preservation of antigenicity of Cx43 was excellent in
high-pressure frozen myocardium (Figs. 1, 4, 5, and 6) and
superior to all previously tested chemical fixation protocols (Fig. 3).
Ischemic Injury
In all age groups, significant increases both in cellular
edema index and in volume-weighted mean mitochondrial
volume were observed, reflecting the ischemic injury
(Table 3).
Qualitatively, Cx43 immunoreactivity was observed in
each of the three compartments defined in the Materials
and Methods section. Gold particles associated with the
specialized membrane compartments were localized in
small gap junction plaques in newborn myocardium
(Fig. 1b). These were observed over the entire cell surface
and interspersed with nonjunctional sarcolemma. In 5and 14-day-old animals, immunoreactive gap junction
plaques occupied larger areas and were usually localized
in close proximity to the intercalated disks (Fig. 1a). Only
few immunogold particles were observed at the plicate
parts of the intercalated disks. Gold particles associated
with the nonspecialized plasma membrane were usually
single particles. Sometimes a higher number of gold particles linked with the free plasma membrane was
detected near gap junction plaques (Fig. 4). AGJs immunoreactive for Cx43 were observed in all age groups and
were frequently located close to intercalated disks (Fig.
5). The mean number of gold particles associated with
AGJs increased during postnatal development. Often, mitochondria were localized in close proximity to gap junction plaques or AGJs. Gold particles were sometimes
observed with mitochondria; however, no systematic
Cx43-labeling of mitochondria was observed (Fig. 6).
The quantitative analysis revealed that the distribution of gold particles in normal myocardium differs between the age groups investigated (total chi-square value:
17.84; P < 0.01). In neonatal myocardium, the number of
gold particles was lower than expected with AGJs and
higher than expected with the nonspecialized membrane
compartment. In 14-day-old rats, the opposite was observed (Fig. 7a).
Ischemia did not significantly influence the distribution
of gold particles in neonatal and 14-day-old rats (total
chi-square values: 7.33 and 10.79, respectively). In 5-dayold rats, ischemia led to a significant difference in gold
1064
mV(Mi) [mm ]
3
CEI
CEI, cellular edema index; mV(Mi), volume-weighted mean volume of mitochondria.
*p < 0.05 towards control (0 minutes of ischemia); y p < 0.05 towards the previous sample. At no point in time did the values significantly differ between the age
groups.
0.10 6
0.07*y
0.65 6
0.31*y
0.87 6
0.06*
0.42 6
0.10*
0.83 6
0.08*þ
0.42 6
0.16*þ
0.76 6
0.08*
0.32 6
0.14
1.01 6
0.09*
0.45 6
0.18*
0.92 6
0.15*
0.36 6
0.14*
0.85 6
0.13*
0.41 6
0.24
0.70 6
0.10
0.25 6
0.08
0.94 6
0.16*
0.46 6
0.10*
0.98 6
0.11*
0.49 6
0.11*
1.17 6
0.14*
0.50 6
0.15*
0.58 6
0.04
0.22 6
0.10
0.79 6
0.1*
0.34 6
0.13
0.86 6
0.05*
0.33 6
0.09*
0.59 6
0.05
0.22 6
0.08
30
15
0
60
45
30
15
0
30
45
60
0
15
5d
0d
Ischemia
(min)
TABLE 3. Stereological data on ischemic injury
14d
45
60
MÜHLFELD AND RICHTER
Fig. 4. Immunogold labeling of free plasma membrane. Next to a
capillary, a gap junction area between two myocytes is visible (thin
arrows). After separation of the membranes (thick arrows), immunogold
particles are found associated with the free myocyte membrane (14-dayold rat, 15 min of ischemia). endo, endothelial cell; cap, capillary lumen.
distribution (total chi-square value: 29.35; P < 0.001)
with increasing gold counts on AGJs and decreasing gold
counts on specialized membrane compartments in the
course of ischemia (Fig. 7b–d).
DISCUSSION
The present study clearly demonstrates that high-pressure freezing and freeze substitution are highly suitable
tools to generate myocardial specimens for immunogold
labeling and electron microscopy. HPF is a relatively new
tool for fixation of biological specimens for electron microscopy. In contrast to plunge freezing, HPF uses a pressure
of approximately 2,000 bar in order to make the water vitrify and prevent formation of large ice crystals (Dubochet,
1995). Since no chemical fixation is used, the ultrastructure is thought to be closer to the living state and antigenicity is remarkably well preserved (Monaghan et al.,
1998). Similar results using HPF and FS were recently
obtained by Rostaing et al. (2004) and Wang et al. (2005).
Our experiences with this method demonstrate an excellent preservation of Cx43 antigenicity in myocardial preparations of postnatal rats combined with a good ultrastructure. The formation of ice crystals that was observed
in several sections can be explained by the fact that before
freeze substitution the frozen specimens were rewarmed
to 908C. It is known that above 1008C ice crystal formation already commences (Dubochet et al., 1991). In general, ice crystal formation was more often found in the
newborn than in older rat heart preparations. A possible
reason for this may be the higher volume fraction of the
watery content of cardiomyocytes, the sarcoplasm, in newborns (Mühlfeld et al., 2005). Moreover, HPF is only possible for very small specimens. Mechanical damage at the
edges of the specimen will occur in every organ under examination; however, cardiomyocytes are extensively interconnected via gap junctions. Therefore, one must emphasize that between biopsy and fixation of a myocardial tissue block, there may be a ‘‘conduction’’ of mechanical
damage from the outer to the inner myocytes of the block.
HIGH-PRESSURE FREEZING OF MYOCARDIUM
1065
Fig. 6. Localization of Cx43 with mitochondria. Not only were mitochondria (mi) observed in connection with AGJs and gap junction plaques (thin arrows), they sometimes even showed immunogold labeling
for Cx43 (thick arrow).
Fig. 5. Immunogold labeling of annular gap junctions. AGJs (thick
arrows) were frequently found in 5-day-old (a) and 14-day-old rat hearts
(b). Note the usual colocalization of AGJs with mitochondria (mi). a:
45 min of ischemia. The AGJ is located near a poorly preserved and labeled gap junction area (thin arrows). b: 60 min of ischemia. Two annular gap junctions are shown in the interior of a cardiomyocyte. Mitochondria with normal ultrastructure (mi) and such showing severe
ischemic injury (smi) were often coexistent within one cell.
In the present study, the myocardium was usually wellpreserved without signs of mechanical distortion within a
depth of 20–75 mm of the tissue block. However, one
should be aware that this range may easily be shifted toward greater distances from the border zone if the size of
cardiomyocytes increases with age or in other species.
The immunogold labeling technique used in the present study provided an excellent method for detection of
Cx43 in postnatal rat myocardium. It should be mentioned, however, that connexin immunogold labeling is
possible with other techniques and may also yield excellent labeling results (Luke et al., 1989; Fromaget et al.,
1992; Gros et al., 1994; Laird, 1996). In a pilot study, we
tested several paraformaldehyde/glutaraldeyhde fixation
protocols followed by cryosubstitution (Fig. 3) and compared them to HPF. As shown, the latter was superior to
aldehyde fixation in preserving myocardial Cx43 immu-
noreactivity. However, if a protocol of chemical fixation
works for immuno-EM, it offers several advantages compared to HPF. For example, perfusion fixation, as performed by Gros et al. (1994) for detection of Cx40, provides
larger blocks of material in which different parts of an
organ can be studied in a comparative manner within the
same preparation. Stereological investigations on such
material allow determination of the reference space and
the experimental and financial effort is less. The choice of
method surely depends on which advantages and disadvantages are more relevant in a specific investigation.
With respect to the application of stereological methods
in the present study, several issues have to be discussed.
First, after removal of the specimens from their carrier,
they were dropped into the embedding capsules without
preferential orientation. Thus, isotropy was assumed
(Stringer et al., 1982). The application of more accurate
methods to obtain isotropic uniform random materials
such as the isector (Nyengaard and Gundersen, 1992) may
only be possible after embedding and requires a second
step of embedding the isectors. Second, no information
about the reference space was available. Therefore, we
only used stereological methods that do not require the
estimation of the reference space (Howard and Reed,
1998). Third, the comparison of the gold labelings by the
method described by Mayhew et al. (2004) served as a
suitable tool for detecting differences in the labeling pattern. However, for a fuller understanding of the mechanisms behind the differences in labeling distribution, further data would be required such as surface density of the
membrane compartments (Mayhew and Desoye, 2004).
To verify our model, it was tested whether alterations
typical of ischemia occur. Indeed, ischemia led to a significant swelling of cardiomyocytes and their mitochondria.
Interestingly, the injury was similarly severe in all age
groups. Given the concept of increased neonatal hypoxia
and ischemia tolerance (Ostadal et al., 1999; Singer,
1999), this result is surprising. However, although there
is a loss of ischemia tolerance in the rat already within
the first 14 postnatal days (Mühlfeld et al., 2005), this
1066
MÜHLFELD AND RICHTER
Fig. 7. Relative distributions of gold particles. Relative distributions
of gold particles associated with specialized membrane (white), nonspecialized membrane (black), and AGJs (gray). The relative distributions for a specific time point and age group were calculated by dividing the mean gold count of a particular compartment by the mean of
the total particle number. The distributions for normal myocardium
(0 min) were significantly different between the age groups (a; see also
Table 2). Neither in newborn nor in 14-day-old rats did a significant distribution change occur in the course of ischemia (b and d). In 5-day-old
rats, ischemia increased a translocation of Cx43 from specialized membranes to AGJs (c). Asterisks mark those compartments that contributed substantially to the intergroup difference.
decrease is rather small and was not detected with the
current methodology.
Cx43 distribution was significantly different among
specialized, nonspecialized plasma membranes of myocytes and AGJs throughout postnatal development. Cx43
that is transported to the nonspecialized membrane via
the Golgi apparatus and then translocated to gap junction plaques is frequently removed via annular gap junctions (Jordan et al., 2001; Segretain and Falk, 2004). Our
results may allow the interpretation that in newborn rat
hearts the new formation and transport of Cx43 to gap
junction plaques exceed the removal. This interpretation
corresponds well with the observed abundance of Cx43
mRNA in newborn rats and the subsequent postnatal decrease in Cx43 mRNA (Fromaget et al., 1990).
Ischemia did not induce Cx43 distribution changes in
newborn and 14-day-old rats. Only in 5-day-old rats was an
increase in Cx43 immunogold labeling found in AGJs with
a decrease of labeling in the specialized membrane compartment. This result is difficult to interpret and we have
no current explanation. In light of the intermediate distribution values of the 5-day-old rats under normal conditions, it could be hypothesized that ischemia induces a response that favors the Cx43 distribution of the older rat
hearts.
It remains to be determined whether age-dependent
distribution differences are related to a different susceptibility of the postnatal hearts to ischemia. In fact, in a
recent study (Vetterlein et al., 2006), we found that ische-
mic preconditioning (Murry et al., 1986) induced a cardioprotection associated with a redistribution of Cx43 from
gap junction plaques to the free plasma membrane, a distribution pattern similar to the one we found in newborn
rat hearts in the present investigation. It is tempting to
speculate that this distribution pattern is related to a
higher tolerance to ischemia since both ischemically preconditioned and newborn rat hearts are more tolerant to
ischemia (Murry et al., 1986; Singer, 1999; Mühlfeld et al.,
2005). Since heterozygous Cx43-deficient mice do not benefit from ischemic preconditioning (Schwanke et al., 2002),
it would be of particular interest whether they show the
phenomenon of a higher myocardial ischemia tolerance in
the newborn. In connection with the phenomenon of ischemic preconditioning, a study by Boengler et al. (2005)
needs to be mentioned. These authors found immunoreactivity for Cx43 in mitochondria of rat myocardium, which
was increased by ischemic preconditioning. In the present
study, mitochondria were often observed in close proximity to gap junction plaques or AGJs and gold particles
were sometimes associated with mitochondria. This may
hint at a role of mitochondria in the function and metabolism of Cx43 molecules or vice versa.
High-pressure freezing and freeze substitution of rat
myocardium can successfully be employed to study the
distribution of the gap junction protein connexin 43 by
electron microscopy. The specimens generated by this
fixation and embedding technique are suitable to perform design-based stereology with respect to both the
HIGH-PRESSURE FREEZING OF MYOCARDIUM
ultrastructure and the immunogold labeling. However,
one should be aware of the problems that may arise from
tissue orientation and lack of knowledge about the reference space.
Cx43 distribution in gap junction plaques, free plasma
membrane, and annular gap junctions undergoes profound changes during postnatal development, which may
have functional significance in the course of myocardial
ischemia.
ACKNOWLEDGMENTS
The authors thank Ms. S. Freese, Ms. H. Hühn, Ms.
S. Kosin, and Ms. S. Wienstroth for excellent technical support, Ms. C. Maelicke for proofreading the English language, and Professor F. Vetterlein for proofreading the
manuscript and discussing the results with us. The present
study was supported by the research program of the Faculty of Medicine, Georg-August-University Göttingen (to
C.M.), and by a grant from the Youth Travel Fund at the
Federation of European Biochemical Societies (to C.M.).
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