(American Journal of Pathology. 1998;153:703-708.)
© 1998 American Society for Investigative Pathology
Porphyrin Loading of Lipofuscin Granules in Inflamed Striated Muscle
Charles R. Kiefer*
,
James B. McKenney
,
Jane F. Trainor
,
Richard W. Lambrecht
,
Herbert L. Bonkovsky§
,
Lawrence M. Lifshitz¶
,
C. Robert Valeri||
and
L. Michael Snyder*
From the Departments of Hospital Laboratories/Clinical
Pathology,*
Pathology,
Medicine
(and The Center for Study of Disorders of Iron and Porphyrin
Metabolism),
Biochemistry and Molecular
Biology,§
Physiology (and The Biomedical
Imaging Group),¶
University of Massachusetts
Medical Center, Worcester, Massachusetts, and The Naval Blood Research
Laboratory,||
Boston University School of Medicine,
Boston, Massachusetts
 |
Abstract
|
|---|
To further the understanding of oxidative effects on inflammation
injury to muscle fiber structure, fluorescent imaging analysis
of human striated muscle tissues from a variety of inflammatory or
postinflammatory etiologies was undertaken in a search for
accumulated coproporphyrin, a red autofluorescent byproduct
of heme biosynthesis that would theoretically be formed under oxidative
insult. Using a differential excitation method of in situ
analysis, porphyrin autofluorescence was detected in intact
fibers within the context of the yellow autofluorescent subsarcolemmal
lipofuscin granules. Relative measurements of porphyrin concentration
in the granules from different patients indicated that the
acute/subacute inflammatory specimens grouped significantly higher than
the more chronic inflammatory and nonpathological specimens. Myoglobin
was also found to be associated with the granules. Myoglobin heme iron
could potentially serve as a Fenton reagent for the intracellular
generation of hydroxyl radicals, which are responsible for the
oxidation of the porphyrinogens. High-performance liquid
chromatography analysis of extracted dense particles revealed
coproporphyrin as the sole porphyrin present. The observation of
coproporphyrin within lipofuscin granules, previously
unreported, suggests that lipofuscin accumulation in striated
muscle may begin under conditions of acute oxidative stress, as
marked by the oxidation of extramitochondrial porphyrinogens that are
immediately incorporated into the granules.
|
Introduction
|
|---|
The myoglobin-loaded red fibers of
striated muscle would theoretically seem to be especially susceptible
to free radical damage in an inflammatory response. Reactive oxygen
intermediates are released by neutrophils and/or monocytes/macrophages
as one class of many nonspecific defense mediators to contain
infection. Inflammatory responses also occur on re-establishment of
blood flow into tissue temporarily deprived of circulation (reperfusion
injury after ischemia). Accumulating evidence suggests that reperfusion
injury to both the microvascular and parenchymal components of muscle
is mediated by reactive oxygen intermediates released by tissue
infiltrating neutrophils.1-3
Inside the fiber, myoglobin
heme iron could conceivably serve as a catalytic source of hydroxyl
radical generation in a Fenton reaction driven by the released
peroxides crossing the muscle fiber membrane (sarcolemma).4
A natural visible indication of oxidative damage within the muscle
fiber might result from the oxidation of extramitochondrial
nonfluorescent porphyrinogens to their fluorescent porphyrin
counterparts, which can be mediated by hydroxyl radicals, as
demonstrated in vitro.5
The attack of hydroxyl
radicals on alkyl carbons is electrophilic, and the predominant
reaction is hydrogen abstraction.6
Thus, hydroxyl radicals
could oxidize the methylene bridges of the porphyrinogens to the
methene bridges that characterize the porphyrins. By way of analogy, in
hepatic cells, iron increases oxidative stress and enhances the
oxidation of porphyrinogens to porphyrins.7
Iron has been
shown to be a major triggering factor in the development of
biochemically and clinically overt porphyria cutanea
tarda,7,8
in which uroporphyrin crystals in hepatic cells
have been localized to regions with dense ferritin granule
deposition.9
Coproporphyrin is the porphyrin type excreted in patients with muscle
damage resulting from ischemia or infarction.10
Reasoning
that coproporphyrin excretion probably follows an acute intracellular
accumulation, we examined biopsied specimens of striated muscle from a
variety of human inflammatory conditions, acute and chronic
(inflammatory myopathies and acute and chronic occlusive vascular
disease of skeletal and cardiac muscle), for evidence of coproporphyrin
accumulations. We also studied heart muscle from a rat model of acute
cardiac ischemia.
|
Materials and Methods
|
|---|
Human Striated Muscle Specimens
Skeletal and cardiac muscle specimens (from biopsy, amputation,
and autopsy), and histopathological diagnoses where indicated, were
obtained through the Division of Anatomic Pathology, University of
Massachusetts Medical Center. For control specimens, normality was
evaluated through hematoxylin and eosin-stained paraffin sections as
well as frozen sections and were defined by findings of normal size
variability and configuration relative to the patient's age; no
specific abnormalities; no fiber degeneration/regeneration, atrophy or
hypertrophy, inflammation, or vasculitis; and no myopathic changes,
including fiber splitting, ring fibers, or fibrosis.
Rat Muscle Specimens
Experimental myocardial infarction was induced in rats by
isoproterenol injection.11
Briefly, a 500-g male Wistar rat
was injected with 100 mg isoproterenol/kg s.c. The animal was
euthanized under halothane anesthesia 24 hours later, and the heart was
excised and immediately frozen in liquid nitrogen cooled isopentane. A
control rat heart was obtained from an untreated animal of the same
weight, sex, and strain. The care and use of the animals was in
accordance with institutional guidelines.
Digital Imaging Microscopy (DIM)
The microscopic methods have been described
previously.12,13
Briefly, specimens were cut in 10
µm-thick cross-sections, adhered to glass slides, air-dried,
acetone-fixed, and mounted in 50% glycerol, 2.5%
1,4-diazabicyclo[2.2.2]octane in 5 mmol/L phosphate buffer, pH 8. For
DIM analysis, the mounted specimen was optically sectioned (at x150)
in 0.25-µm planes using a standard wide-field microscope, and the
optical data were digitized by a cooled charge-coupled device (CCD)
camera into a 310 x 510-pixel format. Point-spread functions
(PSFs) to establish a three-dimensional standard for removing
out-of-focus light were acquired by using tetramethyl
rhodamine-conjugated 0.1-µm fluorescent beads. Dark current images
(DC) to compensate for instrument background were
acquired for both the specimens and PSFs using the same parameters as
for the corresponding specimen images. Flat field images
(FF), acquired with a rhodamine-conjugated
immunoglobulin G (IgG) solution, were used to control for CCD
sensitivity. The measured fluorescent intensity of this intermediate
stage image (I) was calculated from
(I-DC)/FF. Finally, image
restoration (deconvolution) algorithms were used to remove out-of-focus
light from the sections, improving resolution and contrast.
Calculation of Relative Porphyrin Content of Lipofuscin (LF)
Granules
Sectioned muscle specimens were first examined with a filter set
designed to maximally excite both porphyrin and LF (broad-range
excitation filter 390 to 490 nm, 500-nm long pass dichroic extended
reflection filter, and porphyrin emission filter 585 to 635-nm), and
the optical data were captured by a CCD camera. The same field was then
reexamined after changing the excitation filter (420 to 490 nm), which
eliminated that section of the LF excitation range that also excited
porphyrin. Both excitation filters allowed the same degree of light
transmission (80%) within their wavelength ranges. The CCD images were
then restored, aligned, and displayed. Individual granules were
manually segmented from the field and integrated three dimensionally to
determine the total fluorescence intensities for each filter set.
The mean ratio (PE +
fLF)/fLF (described below) was
calculated from a minimum of three granules for each specimen. All
specimens were cut from frozen tissue stored for less than 3 years at
-70°C. The order of capturing the image sets on the CCD camera was
always the same. Both coproporphyrin and LF are relatively
slow-bleaching, LF more so than coproporphyrin. For this reason, data
from the broad range excitation filter, which would include the
porphyrin component, were obtained first.
The expression (PE +
fLF)/fLF is derived from
[I(PE + f'LF +
fLF)]/[I(fLF)],
where I represents fluorescence intensity, PE
represents the full emission of optimally excited porphyrin
(excitation, 390 to 420 nm; emission, 585 to 635 nm), f'LF
represents a marginal emission of optimally excited LF (excitation, 390
to 490 nm; emission, 585 to 635 nm), and fLF
represents a marginal emission of suboptimally excited LF (excitation,
420 to 490 nm; emission, 585 to 635 nm). The assumption is made that LF
fluorescence at 585 to 635 nm resulting from LF excitation at 390 to
420 nm (ie, f'LF) is very small relative to
PE. This is not an unreasonable assumption,
because probable LF fluorophores that are likely to be excited in this
range, the 1,4-dihydropyridine-3,5-dicarbaldehydes, emit at 435 to 465
nm.14
Thus, the original expression reduces to
I(PE +
fLF)/I(fLF), or
(PE +
fLF)/fLF.
Immunohistochemical Detection of Myoglobin within LF Granules
Anti-myoglobin monoclonal antibody M-2-167 (IgG1)15
and anti-
spectrin (1
1) monoclonal antibody bs-4-119
(IgG1)16
were used at 0.1 µmol/L in conjunction with a
cy5-conjugated goat anti-mouse IgG1 (product code M32011, Caltag
Laboratories, South San Francisco, CA). Immunofluorescent intensities
were calculated by computational summation of planar intensities, one
pixel thick, constructed perpendicular to an arbitrary axis through the
granule. The cy5/baseline values derived for each granule represent the
immunofluorescent (cy5) total granule intensity (excitation, 620 to 660
nm; emission, 665 to 695 nm) divided by a baseline autofluorescent
total granule intensity (excitation, 515 to 565 nm; emission, 577.5 to
632.5 nm).
Extraction and Analysis of Porphyrins from Autofluorescent Dense
Particles
In our modification of a previously published
procedure,17
3 g muscle tissue was homogenized with 9
ml of 10 mmol/L phosphate buffer (pH 7.4), 1 mmol/L
ethylenediaminetetraacetic acid, protease inhibitors, and 1% Triton
X-100. After centrifuging at 1,000 x g for 10 minutes,
the supernatant was recovered, recentrifuged, and separated from the
floating lipids by collection from the bottom (tube puncture). It was
then layered over 30% sucrose and centrifuged at 40,000 x
g for 90 minutes. A fraction of the pelleted material was
checked by DIM and found positive for porphyrin and LF
autofluorescence. A proportion of the pelleted material (1g-equivalent
muscle wet weight) was then extracted with the acetone:HCl/ether:HCl
system referenced above to yield hemin-free porphyrins that were dried,
solubilized in 0.1 N HCl, and analyzed by reversed-phase
high-performance liquid chromatography.
|
Results
|
|---|
Localization of Porphyrin Accumulations to Subsarcolemmal Granules
Examinations for porphyrin accumulations were conducted by DIM. In
the human inflammatory specimens, porphyrin-range autofluorescence was
found to associate with subsarcolemmal granule-like structures, often
occurring in clusters (Figure 1A)
. Such
structures were found much less frequently in nonpathological specimens
from individuals at least 6 years old and were topographically simpler
and of less fluorescent intensity (Figure 1, D
versus B).
The structures were not present in a nonpathological specimen from a
1-year-old child (not shown). The structures were found to be prevalent
in a human specimen of acute ischemic cardiac muscle (Figure 2A)
, but were no more prevalent than in
specimens from chronic vasoocclusive cardiac muscle (not shown). In the
muscle of freshly amputated lower legs with chronic vascular occlusive
disease and gangrene, the structures were prevalent, although the
muscle fiber integrity was typically in poor shape (not shown). Similar
structures were observed in rat acute ischemic cardiac muscle (Figure 2B)
but not in controls (not shown). The apparent correlation of the
number and size of these structures with age, and their subsarcolemmal
location, suggested their association with muscle LF granules, the
subsarcolemmal yellow autofluorescent age pigment.
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Figure 1. Red fluorescent granules in human skeletal muscle. Frozen muscle biopsy
specimens were sectioned and analyzed by DIM. To induce and detect
maximal fluorescence of accumulated porphyrin, excitation
(400 to 410 nm) and
emission (585 to 635 nm)
filter ranges were chosen on the basis of excitation and emission scans
of soluble purified coproporphyrin III
(Porphyrin Products, Logan,
UT). In A and C, single restored
optical planes (150x)
are presented to better demonstrate the location of the autofluorescent
granules (arrowheads) within the structural context of the
muscle fibers. In B and D, segments of A and
C, respectively, are presented in their three-dimensional
restored forms. The black/white context is reversed to better visualize
the topography of the granules. A: Specimen from a 43-year-old
male with dermatomyositis (bar = 5
µm). B: Three-dimensional restoration
of a segment of A. C: Specimen from a normal
24-year-old male. D: Three-dimensional restoration of a segment
of C.
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Figure 2. Red fluorescent granules in cardiac muscle. Frozen specimens of cardiac
tissue were examined
(150x) for the presence
of accumulated porphyrin as described in Figure 1
. A: Autopsy
specimen from a 29-year-old female (left
ventricle infarction associated with disseminated intravascular
coagulation). B: Rat cardiac muscle
taken 24 hours after induction of acute cardiac ischemia by
isoproterenol.
|
|
Porphyrin Accumulations as a Function of Inflammatory Status
To determine whether porphyrin accumulation was increased in
inflamed muscle tissue, we sought to measure, in situ, the
porphyrin content per unit volume of LF. LF fluorescence is best
measured by using a broad-range excitation filter (390 to 490 nm) with
a 515-nm barrier filter.14
The emission peak for LF in
muscle is within the 510 to 530-nm range,18
although its
fluorescence extends into the porphyrin range.14
Porphyrin
fluorescence peaks at 600 to 630 nm and is characteristically
excited at wavelengths near 400 nm.10
Thus, excitation of
porphyrin would also excite LF to some extent, the fluorescence from
which would overlap somewhat that of porphyrin. A relative measure of
porphyrin within an LF context could be calculated from porphyrin-range
fluorescence intensities at two excitation ranges. The range 390 to 490
nm would maximally excite both porphyrin and LF, and the range 420 to
490 should excite a major fraction of LF but very little of porphyrin.
For any given granule, the ratio of these two fluorescence intensities
could be expressed as (PE +
fLF)/fLF (a ratio of porphyrin to
lipofuscin spillover), where PE represents fluorescence due
to porphyrin, and fLF represents the suboptimally
excited, marginal LF fluorescence within the porphyrin emission range.
Using this differential excitation method, Table 1
shows significantly elevated ratios for
specimens of acute inflammatory skeletal muscle and acute ischemic
cardiac muscle versus those found in more chronic
inflammatory/ischemic or nonpathological striated muscle, indicating
porphyrin loading of LF granules during acute inflammatory episodes. To
determine whether the distribution of porphyrin within the LF granule
was either homogeneous or shell-like, cross-sectional analysis of
individual granules from the adult dermatomyositis specimen was
performed. The Gaussian-like curves of both signals (Figure 3)
indicated fairly homogeneous
distributions (a predominantly bimodal curve would have indicated a
shell-like distribution). It is noteworthy that loading of porphyrin
into LF granules can occur rapidly under conditions of experimental
acute cardiac ischemia (Figure 2B)
.
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Figure 3. Cross-sectional analysis of porphyrin/LF granules for distribution of
fluorescent signals. DIM data from dermatomyositis skeletal muscle
specimen (Table 1
, specimen
1) was collected for porphyrin plus partial LF
fluorescence ("P": excitation 400 to 410 nm;
415-nm long-pass dichroic; emission, 585 to 635
nm) and for suboptimal LF fluorescence exclusive
of porphyrin ("LF": excitation, 420 to 490
nm; 500-nm long-pass dichroic extended reflection; emission, 520 nm
long-pass barrier filter). Presented here is a
single granule from the field that was computationally analyzed for
fluorescence intensity of each signal in sequential planes 1 pixel
thick (0.187 µm)
constructed perpendicular to an arbitrary axis through the granule.
Results are presented as planar fluorescence intensity
(normalized to 1.0 for each
signal) versus position
(µm) along the granule
axis.
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Identification of Porphyrin Type
LF granules exhibit a density of 1.140 to 1.178
g/cm3.19
In an attempt to isolate porphyrins
associated with these granules, chronic vaso-occlusive muscle samples
from both amputated leg and autopsied heart (Table 1
, specimen 7) were
solubilized and an autofluorescent-dense (>1.132 g/cm3)
particulate fraction (checked by DIM) was extracted using a solvent
system designed to isolate hemin-free porphyrins.17
The
isolates were then examined for spectral excitation and emission
characteristics and checked for high-performance liquid chromatography
retention time in comparison with porphyrin standards. By these
criteria, the isolates were found to be the same from both skeletal and
cardiac tissue. The extracted porphyrin exactly resembled
coproporphyrin III in both spectral characteristics
(
excite = 406.0 nm; emit = 604.8 and
607.9 nm) and high-performance liquid chromatography retention time
(single peak, 10.80 minutes, versus 10.78 minutes for
coproporphyrin III standard).
Presence of Myoglobin within the Porphyrin/LF Granules
The autofluorescence of LF granules poses obvious special
problems for the immunofluorescent detection of specific antigens of
interest, requiring a quantitative approach that controls for LF
fluorescent background within the fluorescent emission range of the
immunospecific signal. To determine whether myoglobin (and presumably
heme iron) associated in situ with the porphyrin/LF
granules, we set up a quantitative immunofluorescent DIM assay. Mouse
monoclonal antibodies of the same isotype (IgG1) were used to detect
the presence of myoglobin and the absence of spectrin (11
isoform), the latter expressed only in red cells and thus a negative
control. Because the overall autofluorescence of the granules was less
intense in the far red region, despite the contribution of
coproporphyrin, we chose cy5 as the fluorescent reporter conjugated to
the second antibody, an affinity-purified antimouse IgG1. To normalize
the immunofluorescent data collected from eight individual granules per
assay, we calculated the ratio of immunofluorescent intensity observed
with the cy5 signal divided by that observed for a baseline intensity
closer to the autofluorescent peak of LF. The latter was accomplished
by using a tetramethyl rhodamine filter set, for which the excitation
range does not overlap that of cy5. By such an analysis (Table 2)
, we determined that myoglobin was
associated with the granules (P < 0.01,
t-test).
|
Discussion
|
|---|
Although it has generally been understood that LF formation in
striated muscle is a gradual, age-related process,14
the
data presented suggest that porphyrin-loaded LF granules may also form
within an acute oxidative context. Although conclusions cannot be
directly extrapolated to humans from the experimental rat model (Figure 2B)
, the relative porphyrin content of LF granules in human skeletal
muscle (Table 1)
, as well as their prevalence (Figure 1, A
versus C), directly correlates with recent inflammatory
history. The association of myoglobin with the granules (Table 2)
supports the hypothesis that heme iron-catalyzed generation of hydroxyl
radicals may play a role in the oxidation of cytoplasmic porphyrinogens
and their loading into the granules.
|
Acknowledgements
|
|---|
We thank Dr. Eric Dickson, Department of Emergency Medicine,
University of Massachusetts Medical Center, for technical expertise
with the rat myocardial infarction model.
|
Footnotes
|
|---|
Address reprint requests to Dr. Charles R. Kiefer, UMass Memorial Health Care, University Campus, Department of Hospital Labs/Clinical Pathology, 55 Lake Avenue North, Worcester, MA 01655-0220. E-mail:
charles.kiefer{at}banyan.ummed.edu
Supported by the US Navy (Office of Naval Research Contract No. N00014-79-C-168, with funds provided by the Naval Medical Research and Development Command), and the US Public Health Service (National Institutes of Health grant DK 38825 to HLB).
Accepted for publication May 30, 1998.
|
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Top
Abstract
Introduction
