Ultrastructural Characteristics and DNA lmmunocytochemistry in Human lmmunodeficiency Virus and Zidovudine-Associated Myopathies G. PEZESHKPOUR, MD, ISABEL ILLA, MD, AND MARINOS C. DALAKAS, MD Electron
microscopic
immunodeficiency receiving
zidovudine
patients AZT.
features (AZT)
specimens
structures,
myopathy
showed
inflammation,
files. One untreated
13 human
with biopsies
who were
disorganization
along with a varying
vacuolization,
from
patients who had myopathy
were compared
with HIV-induced
All
of muscle biopsies
(HIV)-positive
degree
from five
not treated
with
of the myofibrillar
of nemaline
and endothelial
and all AZT-treated
while
(rod)
bodies,
tubuloreticular
patients
pro-
had cytoplasmic
bodies, which in the latter were abundant, large, and irregular. untreated
patients
fibers,
muscle dothelial
had a peculiar
with
numerous
osmiophilic
tubuloreticular
cells and brisk inflammation
matoid
cells. The AZT-treated
tochondria
group
that complemented
seen by light microscopy. of mitochondria, liferation
There
or disorganization
profiles
that included
guished
showed
patients
abnormal
mitochondria
with AZT-associated
untreated
H[IV-induced
to single- and double-stranded
of mitochondrial
DNA. Although common
DNA
the myopathies
myopathologic reduced,
Immunocytochemistry DNA
revealed
using severe re-
with HIV and AZT share
the mitochondrial
patients.
the structural
;are probably
a DNA chain terminator
polymerase,
distin-
those with
with the normal
microscopy
22:1281-1288.
from
compared
Zidovudine, y-DNA
inclu-
of the electron
associated
features,
are unique to the AZT-treated is specifically
proliferation
that readily
myopathy
myopathy.
mi-
red fibers
of their cristae. Paracrystalline
antibodies duction
abnormal
in size and shape and pro-
sions were seen in one patient. Blind re-examination micrographs
in the en.
of ragged
was subsarcolemmal
variation
Two of the
lymphoplas-
had ubiquitous
the presence
with marked
destruction
nuclear
abnormalities
Since mitochondrial changes
noted
associated with mitochondrial
DNA
on electron dysfunction.
that inhibits the mitochondrial
is toxic to muscle mitochondria.
This is aUS government
work. There
HUM PATHOL are no restric-
tions on its use.
(:linicxl signs of myopathy characterized by muscle weakness and elevation of serum creatine kinase frequently O(‘CLII‘in human immunodeficiency virus (HIV)____
positive patients.‘.’ ’ The muscle hiops) sprGmens from these patients show a variety of ft’c\turcs, including phagocytosis. degeneration or necrosis of ~uusclr fibers, endomysial or perimysial iriflammation. cytc~plasniic bodies, and nemaline (rod) bodies. Following the introduction of 4dovudine (AZT) for thy treatment of the acquired immunodeficiency syndrome (AI 1x5). t hc number of HlV-positive patients with myopathic symptoms has increased.‘i.7 Zidovudine has burl implicated as the cause of the myopathy because these symptoms generally improve when AZT is dis~otltirlu~d.“‘~‘” In an effort to distinguish AZT-associated myopathy from that dne to HIV alone, we have recently shown in a comparativr analysis that AZT. but not HIV. is associated with specific changes in the muscle Illitoc.hondI.ia that appear on light microscopy as “ragged red” fibers.“.‘H We suggested that these changes art’ due to inhibition by the AZT of the I~USC le mitc~c!~oIldrial yDNA polymerase. 7.‘7.‘HOur findings were confirmed b! some investigators”‘,“’ and disputed by others. !’ Establishing that AZT is toxic to muscle mi;c,(:h(,ndr-ia is important for the following reasons: (1) A%T is widelv used not only in the treatment of AIDS, but also in’ HIVpositive patients without AIDS; (2) this information can provide morphologic clues to distinguish the AZT-induced myopathy from that causrd hy HI’1’. and therehjprovide guidance for therapeutic dksiona; and (3) it may help us understand how DNA chain ternlinators, such as AZT, can play a causative I-c& in the induction of mitochondrial myopathies. For these reasons, we have extended our previous observations at the ultrastructural level by comparing the electron microscopic features of thy muscle biopsies from 13 HIV-positive patients whc I cxpcrienced II~Yapathy while receiving AZT (AZT-My) with the findings from hve patients with HIV-associatt~l myopathy who did not receive AZT (HIV-My). (1hanges in the muscle mitochondria DNA were also examinedl hv immunocvtochemistry. We describe sp4fic structlural changes associated onlv with AZT, but not with HIV. MATERIALS
1281
AND METHODS
HUMAN PATHOLOGY
Volume 22, No. 12 (December
elevation of the serum
creatine kinase level. All patients were male homosexuals with the exception of one female drug abuser. The patients in whom electromyography was performed had evidence of myopathic changes, as previously reported. ‘-” The muscle biopsy specimens were fresh-frozen in isopentane cooled to -70°C and processed for muscle enzyme histochemistry, including staining with modified Gomori trichrome,“,28
Immunocytochemical studies were performed to determine changes in mitochondrial DNA in the muscle biopsy specimens from all patients. The first of three serial, 4 pmthick frozen sections was stained for modified Gomori trichrome” and the third for ATPase, pH 9.4, while the second section was processed for immunocytochemistry as follows: sections were fixed in 4% formaldehyde containing 0.1 mol/ L CaCl,, pH 7.0, for 1 hour at room temperature, then dehydrated in a graded series of ethanol concentrations (50%, 70%, 80%, and 90%) for 5 minutes and in 100% ethanol for 15 minutes in the last dehydration step. After washing three times in phosphate-buffered saline for 5 minutes, the sections were incubated for 2 hours with a monoclonal antibody against both single- and double-stranded DNA (Chemicon #MABOJ 1, Temecula, CA) diluted 1: 10 in phosphate-buffered saline containing 1% bovine serum albumin. The sections were subsequently washed three times in phosphate-buffered saline, and incubated for 1 hour at room temperature with goat antimouse rhodamine-conjugated immunoglobulin G (IgG) diluted 1:40 in phosphate-buffered saline containing 1% bovine serum albumin. The sections were then washed, mounted, and examined with a Zeiss photomicroscope equipped with epi-illumination. Staining within the muscle fibers and the myonuclei was compared in the specimens from HIV-positive patients treated with AZT and those not treated with AZT. Differences in the intensity of the endomyofibrillar staining between types I and II fibers were assessed in serial sections stained with ATPase. Changes within the ragged red fibers were assessed on serial sections stained with the trichrome. Controls for the
Another specimen from the biopsied muscles was fixed in 2% buffered glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, for 30 hours and processed for electron microscopy. After three 15-minute washes in the same buEer. these specimens were minced and postfixed in 2% buffered osmiumtetroxide for 2 hours, dehydrated in graded alcohols, cleared in propylene oxide, and embedded in Poly/bed 812 (Polysciences, Inc, Warrington, PA). One micron-thick sections from a minimum of five blocks per patient (the number of blocks examined ranged from five to 10 per patient) were cut with an ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a Zeiss 9 electron microscope. To assess features unique to the AZT-treated group, after thoroughly reviewing the blocks for abnormal structures, viruses, cytoplasmic bodies, rods, and mitochondrial changes, one of us (G.P.) photographed representative areas from all the blocks, with special attention to mitochondrial accumulations. Subsequently, the sets of photographs from each of the 18 patients were coded and reviewed independently by two of us (G.P. and M.D.) for the presence and frequency of all the abnormal structures mentioned above. TABLE 1. Histologic
.4ge t’;ltient No.
(yr),‘Scx
“H/M
?4,fM .W/hI %,‘hf
1991)
and Electromicroscopic Features of Muscle Biopsies of Human lmmunodeficiency Virus-Positive Patients With Myopathy
Dwation of AZT Therapy (mo)
Main Hi\tntogic Findings
PM PM PM; C:B Rods; type
%RKF*
III< reased No.
Six Variatim
PS
Crt~~ptasrnic Bodies
I 0.3 0.
I
PM
1:4/F 34/M .l!)/hl 43/M .58/M 41/M 47/M 32/M -12/M N/M 44/M 28/M 36/M
PM; PM; PM; PM; PM: PM; PM; PM; PM: PM; PM; PM; PM;
CB CB CB CR CR (:B CB CR CR CR C:B Cl3 C:H
TR PI-ofites
_ _ ++t
+
IJ
f%xl woptl~ “X/h1
Rods
,Nec~-otic 01 Ikgenerating Fibers
_
4.4
8.3 8 19.7 x.7 28 48 R-1 4.4 x I2 7 I4
+ + t + + + + + + + + + +
+ + + + + + + + + + + + +
_ _ _ _ _ _
+ _ _
+ +++ ++ t+t + + + t + _ +t
Abbreviations: PM, polynryositis charactc%zed by varying degrees of endomysial inflammation, degenerating cytoplasmic bodies; PS, paracrystalline structures; TK, tubutoreticular. * Percentage of “ragged-red” fibers.
1282
t
t++ ++ t-+t + ++ t ++ + ++ +t ++ ++ tt
fibers, and phagocytosis;
_ _ _ _ _ _ _ + + CB.
ZIDOVUDINE-INDUCED
MITOCHONDRIAL
MYOPATHY
(Pezeshkpour
et al)
FIGURE 1. Electron micrograph of a muscle biopsy specimen lrom a patient with HIV-induced myopathy showing nemaline (rod) bodies within a myoftber. (Magnification ~4,182.)
iltimlrlic~c.\itochc.lnic pt-inmr-y
;;nt
c.onc.cntl‘atioli. 11om patients Sqrc
aI staining
iht )dy with
mouse
IXseasc with
cptr0111~)
wntrols
known and
othu
included lg(;
of
substitution the
same
included
mitochondrial myoparhies
biopsy myopathir\
with
01
subclass
the and
specimens (Kearns-
deqmx~tin~
fi-
kr-\.
RESULTS With light microscopy, the muscle specimens of all AZT-treated patients, but none of the five untreated ljatients, had unique morphologic features characterized by abundant ragged red fibers suggestive of abnormal Initochondria, as previously described. “7.‘HThe number (bf ragged red fibers in the AZT-My group ranged from -$.4% to 4-872 (mean, 14.2%); in the HIV-My group, the slumber I anged from 0% to 1 YO(mean, 0.3%). The latter was similar to the number of ragged red fibers counted in 25 muscle biopsy specimens from patients with inIlammatory myopathies, which ranged from 0% to 2.9% (mean. O.f%).” All the specimens, including those with abnormal mitochondria from the AZT-I reated patients, had histologic features of inflammatory myopathy, as seen in polymyositis, characterized by varying degrees t )f endomvsial inflammation with necrosis and phagocytosis of inuscle fibers. Rod (nemaline) bodies” and cytoplasmic bodies were numerous (Table 1). Necrotic fibers wit11 vacuoles were prominent in the AZT-treated patients. Electron microscopic examination of the muscle biopsies f’rom patients with HIV-My who never received ,AZT showed inflammation, with a predominantly interstitial chronic inflammatory cell infiltrate composed of macrophages, plasma cells, and a few lymphocytes. Infiltration of the myofibers by these cells was not a prominent finding. Additional findings included tubuloretic.-
1283
ular profiles, as previously described,’ in the endothelial cells of one patient, nemaline (rod) bodies in another patient (Fig 1 ), occasional small cytoplasmic bodies, and mild increase in myofiber fat content. Patient no. 3 (Table 1) showed a severe necrotizing myopathy with large numbers of necrotic fibers and a peculiar osmiophilic degeneration of the sarcoplasm (Fig 2). Mitochondria md other membrane-bound elements of the cell, transverse tubules, sarcoplasmic reticulum, and the thick and thin filaments were not affected. Likewise, the neuromuscular junctions, both at the presynaptic and postsynaptic sites, the sarcolemma. and the ‘basement membranes appeared normal (Table 1). No viral particles were noted. In patients with AZT-My, the most striking abnormality was a marked proliferation of mitochondria, especially in the subsarcolemmal regions, with extreme variations in the mitochondrial size and shape, as illustrated in Fig 3. The mitochondria ranged from 1 pm (normal) to 12 to 15 pm, with changes ranging from marked proliferation to disorganization. disruption, loss of concentric arrangement of the cristae, and occasionally small paracrystalline structures (Table 1). One patient had typical paracrystalline inclusions (Fig 4). I’rotein-lipid complexes within the mitochondrial matrixes were easily identified. In addition to the mitochondrial abnormalities, muscles from the AZT-treated patients showed a large number of cytoplasmic bodies of different sizes, either isolated or in small groups, that commonly appeared as conglomerates with a stellate shape (Fig 5). Other than their size, however, the ultrastructural appearance and organization of these cytoplasmic bodies were not different from those occasionally noted in muscle biopsies of HIV-My patients or those seen in other myopathies. Nemaline (rod) bodies, more easily identified in a few
HUMAN PATHOLOGY
Volume 22, No. 12 (December
1991)
FIGURE 2. Electron micrograph of a muscle biopsy specimen from a patient with HIV-induced myopathy showing osmiophilic destructive changes in a myoftber. (Magnification ~4,131.)
muscle fibers with light microscopy, were seen in one of the AZT-treated patients (Table 1). Myofibrillar disorganization, atrophy, and Z disk streaming were frequently seen. Degenerating and necrotic fibers with pyknotic nuclei and condensed cytoplasm were often present. Of interest was the marked paucity of regenerating cells. Tubuloreticular profiles in the endothelial cells were noted in two patients (Table 1). With immunocytochemistry, a distinct punctate staining pattern representing immunostainable mito-
chondrial DNA was noted within the myofibrils of the normal muscles, the muscles from patients with other myopathies, and the muscles from patients with nonAZT-treated HIV-My (Fig 6A). The myonuclei were strongly positive (Fig GA). In the muscle from patients with Kearns-Sayre syndrome, there was increased immunostaining throughout the myofibrils, with strongly stainable mitochondrial DNA within the subsarcolemmal mitochondrial accumulations of the ragged red fibers (Fig 613). In contrast, the muscle biopsies from the AZT-
FIGURE 3. Electron micrograph of a muscle biopsy specimen from a patient with AZT-associated myopathy showing abnormal mitochondria with marked variations in size and shape and internal disorganization of cristae. (Magnification x 14,580.)
1284
ZIDOVUDINE-INDUCED
MITOCHONDRIAL
MYOPArHY
(Pezeshkpour
et al)
FIGURE 4. Electron micrograph of a muscle biopsy specimen from a patient with AZT-associated myopathy showing proliferation of subsarcolemmal mitochondria with occasional paracrystalline inclusions. (Magnification :x 14,760.)
treated
01’ thc intpatients showed severe reduction munostainable mitochondrial DNA cot~~pared with tttc. normal nuclei DNA (Fig 61) and F). ~‘I~cw c~ltangc*~, which were more severe in the patients with ad~xnct~cl myopathy (Fig 6F compared with Fig 61)), W~TCpresent in all the fibers, including thr ragged t-cd fibers, as sec’n in serial sections stained with trichrome (Fig fi(; and 1;.) and with immunocytochemistry (Fig tin a11d (il;). ‘I‘ht reduction of the immunostainable tnitoc.hotttlt~i~tl I)N,,I was more pronounced in type 1 fibers. wtiic-lt arc’ ric.11
FIGURE 5. Electron micrograph of a muscle biopsy specimen from a patient with AZT-associated myopathy showing numerous single or stellate cytoplasmic bodies. (Magnification ‘. 4,182.)
1285
itt ttti~oc~ttc~tttlt-i;1.as deduced front serial s8t.c.tion5 slained \vith .\‘l‘l’acc (data not showni. DISCUSSION PVC’II \vliett Ilr~ t~lcOt~ott ttticror;c-opic- tindittq, I)littdl\~ t-c~\it~wed. rr~~3led that thr. tttuac~les t rotn 1tic lxtlic.ttts l+illt ;\%‘I‘-hl\ sh o\cetl tlis~iticr f’catttt-es 110t ~m3ml iii lhc, HI\‘-kl\ patients. ‘1%~ tttain distin~~ishitt~
FIGURE 6. (A) Transverse frozen section of a muscle biopsy specimen from a patient with HIV-associated myopathy who did not receive AZT, immunostained with antibodies to DNA and rhodamine-conjugated anti-IgG. Note the normal punctate pattern in the intermyotibrillar mitochondria. The fibers with a less bright punctate pattern are type II ftbers, based on serial sections stained with ATPase (not shown); the nuclei are brightly stained. (B) Transverse section of a muscle biopsy from a patient with Kearns-Sayre syndrome immunostained with anti-DNA antibodies showing strong immunostaining pattern in the endomyofibrillar mitochondria and the subsarcolemmal mitochondrial accumulations within the ragged red fibers (arrow). (C-F) Serial sections of muscle biopsy specimens from two patients treated with AZT, one having mild myopathy (C and D) and one having advanced myopathy (E and F), stained with trichome (C and E) and antibodies to DNA visualized with rhodamine (D and F). Punctate staining of intermyofibrillar mitochondria is uniformly reduced within the muscle fibers, including those fibers that show an accentuated intermyofibrillar membranous network (probably ragged red fibers: arrows, C and E). The reduction is prominent in the patient with advanced myopathy (F). (Magnification X560.)
ZIDOVUDINE-INDUCED
MITOCHONDRIAL
leature was the presence of mitochondria that were abnorn~al in number, size. shape, and structure. These findings complement our previously reported light mimicroc.roscopic studies ” and indicate that electron scopic and light microscopic assessment of the muscle t)iopsy is a reliable diagnostic tool to determine whether the myopathy is related to the administration of AZT. Llnlike the occasional abnormal mitochondria inc%ientally present in the muscle biopsies of patients with inflammatory myopathies and inclusion body myositis, thr changes seen in AZT-My were ubiquitous and occ.urred in varying degrees in the majority of the mitoc~hondria. These changes were also, unlike those associated wit11 ~nusclc fiber necrosis or phagocytosis, characterized by occasional dark, spherical mitochondria ‘ with dense crist,te.““.?’ In AZT-My, there was proliferation of miu)chondria of variable sizes and shapes, with extreme disorganization or loss of their cristae and occ&onal paracrystalline inclusions resembling those seen in patients with mitochondrial encephalon~yopathies.g5~“” C:haracteristically, no such changes were noted in HIVML- in spite of‘ the degenerative, phagocytic, and necrotizing nasture of the myopathy. This indicates that the abnormal rnitoc-hondria are not due to HIV alone, but rather due to the effects of AZT. Zidovudinc is a DNA chain terminator that inhibits the mitoch~ondrial y-DNA polymerase and interferes with the protein synthesis.“,“x Structurally, abnormal mitochondria such as those described above may not function normall!*. Although there is no specific correlation hetwern structure and function of altered mitochondria. the observed changes may cause uncoupling or a defect in substrate utilization and the respiratory chain system. Our preliminary data in rats suggest that AZT causes early uncoupling. affecting the complex IL of the mitc&ondrial respiratory chain.” Evidence that these Illitol.-horldria may function abnormally, resulting in rt&~etll protein synthesis, is supported by our inltll~lno~ytoc~llenlic’al staining, which showed severe reduct& of‘ mitochondria DNA in contrast to normal nuclear DPiA. not only within the ragged red fibers, hut also within the rest of the fibers (Fig 6). Since individual mitochondria contain multiple DNAs, with an average of two to 10 mitochondrial DNA per organelle,“’ the diminished staining e\‘en within the ragged red fibers suggests a generalized reduction of mitochondrial DNA, which probable hate fdiktl to replicate successfullv. This appears to be ‘iI1 contrast to the ragged red fibers seen in patient?, with Kearns-Sayre syndrome, in whom the k~~ow~~ nlito~t~ol~clrial deletions”’ probably do Ilot affect the DNA rc~plicatiori, as evidenced by the strong subaarcolemntal staining we noted immunocytoc~lemicall~ (Fig CR). ‘l‘hese tindings arc also consistent with out Inolecular 1)NA siudies in the muscle biopsies f’rom the sine XZT-treat4 patiellts. which showed up to 8OYa rtduction of the mitoc~hondrial DNA as assessed hy the Southern blotting analysis and scanning of the blots.‘” M’hethel- the changes i;lduced by AZT are muscle-specific or alyo occur to some extent in other organs is unknown. Muscle (~11s may be more susceptible to these nlitochoiictrial chanys because the)- are long-lived, rnultillrl~l~,;lt~~l stncvtia that, unlike the cells in other 1287
MYOPATHY
(Pezeshkpour
et al)
organs such as the liver or kidneys, do not have