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Aggregates of tubules in human cardiac muscle cells
Journal
of Molecular
and Cellular
Aggregates
Cardiology
(19741,
of Tubules
in Human
BARRY J. MARON Cardiology
Branch
6,249-264
AND
VICTOR
and the Section of Pathology, .National of Health, Bethesda, Maryland (Received
30 July
1973,
Cardiac
Muscle
Cells
J. FERRANS
Heart and Lung Institute, 20014, U.S.A.
and accepted 4 October
.National Institutes
1973)
B. J. MARON AND V. J. FERRANS. Aggregates of Tubules in Human Cardiac Muscle Cells. Journal of Molecular andCellular Cardiology (1974) 6,249-264. Two types ofaggregated tubules derived from, and continuous with, elements of sarcoplasmic reticulum were observed in degenerated cardiac muscle cells from patients with cardiac hypertrophy. Aggregates of the first type, found in 2 patients, were composed oflong, parallel tubules which measured from 500 to 1130 A in diameter and showed different degrees of organization. In some of these aggregates the tubules were irregularly arranged and were closely associated with large masses of abnormal 2 band material to which thin myofilaments were attached. In other instances, the tubules formed highly regular, hexagonally arranged aggregates; the spaces between these aggregated tubules contained thin myofilaments and small amounts of Z band material. Cells in which these aggregates were present showed advanced degenerative changes characterized by disarray of myofibrils, absence of T tubules, and selective loss of thick myofilaments. It is concluded that the formation of these aggregates of tubules derived from the sarcoplasmic reticulum is a degenerative change related to the inability of the sarcoplasmic reticulum to form meshworks around abnormally constituted myofibrils, or terminal cisterns around T tubules. Aggregates of the second type, found in one patient, were formed by long, straight, irregularly arranged tubules that measured 500 to 830 A in diameter and were not associated with myofibrillar components. These aggregates are considered to represent an unusual type of proliferation of the sarcoplasmic reticulum in cells having normally arranged contractile elements. KEY
WORDS:
degeneration;
Cardiac Aortic
hypertrophy; regurgitation;
Ultrastructure; Hypertrophic
Sarcoplasmic cardiomyopathy.
reticulum;
Myofibrillar
I. Introduction The sarcoplasmic reticulum of cardiac muscle cells is a specialized network of intracellular tubules which are connected with the outer nuclear membranes and form a meshwork around each myofibril. The sarcoplasmic reticulum has specialized areas, known as terminal cisterns, which are closely approximated to the extracellular environment by virtue of their apposition to the plasma membranes. The areas of apposition occur either near the cell surfaces or, deeper in the cell, in relation to portions of the transverse tubular system and to the Z bands of the myofibrils [I, 531. Because of its important role in cardiac contraction, numerous studies have been made of pathologic alterations in the sarcoplasmic reticulum of cardiac muscle
250
B.
J.
MARON
AND
V.
J.
FERRANS
cells. Changes observed include swelling or dilatation of the tubules, which occurs as a non-specific finding in a variety of conditions [25, 321, and increased amounts of morphologically normal tubules, which have been reported in cardiac hypertrophy [47j. The purpose of this communication is to describe alterations of sarcoplasmic reticulum that have not been previously reported in myocardium. These alterations consist of two different types of aggregates of tubules formed by modified components of sarcoplasmic reticulum in degenerated cardiac muscle cells of patients with cardiac hypertrophy.
2. Materials
and
Methods
Observations described in this communication are based on study of myocardial biopsies obtained at operation from the left ventricular apex in 14 patients with aortic valvular disease and from the left ventricular posterior wall in 10 patients with hypertrophic cardiomyopathy (asymmetric septal hypertrophy) . All tissues were fixed in cold 3% glutaraldehyde in 0.1 M-phosphate buffer, pH 7.2. After washing with several changes of cold 5% sucrose in 0.1 M-phosphate buffer, pH 7.2, the tissues were postflxed with 1 y0 osmium tetroxide in Millonig’s phosphate buffer, pH 7.2, dehydrated with a graded series of ethanols and propylene oxide and embedded in Maraglas [27]. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed with either an RCA EMU-3G or JEOL 1OOB electron microscope.
3. Results The morphologic characteristics of the two types of aggregates described in this study are summarized in Table 1. The first type of aggregated tubules was observed in two patients: patient No. 1 was a 44-year-old man with severe aortic regurgitation; patient No. 2 was a 24-year-old man with non-obstructive asymmetric septal hypertrophy (hypertrophic non-obstructive cardiomyopathy) and pulmonary sarcoidosis. The second type of aggregate was observed in a 46-year-old man (patient No. 3) with aortic regurgitation. Aggregates of either type were not found in the other 21 patients. Aggregates of tubules of the first type occurred only in cardiac muscle cells showing features of severe degeneration (Plates 1 and 2). These features were: loss almost complete absence of myosin filaments; of normal myofibrillar structure; clumps of abnormal Z band material; numerous actin filaments, both free in the sarcoplasm and attached to the abnormal Z bands; markedly increased numbers of tubules of sarcoplasmic reticulum, which were focally dilated; unusually small mitochondria; absent T tubules, and thickened basement membranes. In sections cut parallel to the longitudinal axes of the myofibrils, the abnormal Z band
between
500-875 560-l 130 500-830
Overall tubular diameter (4
characteristics
distance
Hexagonal Hexagonal Irregular
1 2 3
* Center-to-center
Arrangement of tubules
1. Morphologic
Patient No.
TABLE
adjacent
50-65 65 -
of
in cardiac
Diameter intertubular filaments (4
of tubules
tubules.
660-830 900-1200 -
Inter-tubular distance* (4
of aggregates
cells
50-65 65 60
Thickness of tubular wall (4
muscle Size of tubular aggregates Cd
5200 0.7 x 1.2 9000 0.7 x 1.6 8500 1.4 x 2.5 _-_-___~
Maximum length of tubules (4
50 65 35
Maximum no. tubules per aggregate
5 7 4
Maximum no. aggregates per cell
252
B.
J.
MARON
AND
V.
J.
FERRANS
material appeared to be traversed by filaments that were arranged in a regular, parallel fashion, measured 65 A in diameter and were separated from each other by distances of approximately 100 A (Plates 3 and 4). Because of their association with Z band material, these filaments appeared more electron dense in the Z band areas than elsewhere. Such filaments corresponded morphologically to the thin (actin) myofilaments and extended as much as 1.5 pm from the centers of the abnormal Z bands, but were not associated with thick myofilaments (Plates 3 and 4). In sections cut perpendicular to the longitudinal axes of these filaments, the abnormal Z band material appeared finely fibrillar and often exhibited a square lattice type of substructure (Plate 5). Adjacent accumulations of Z band material with attached thin myofilaments were often oriented obliquely or perpendicular to each other, indicating a loss of normal myofibrillar alignment (Plates 2 and 3). In some areas, the thin myofilaments attached to the masses of Z band material were tangled and disorganized (Plates 6 and 7) ; in other areas, the aggregations of thin myofilaments appeared to be associated with little or no Z band material (Plates 1 and 2). The tubules constituting the first type of aggregates (Plates 1 to 3, 5 to 11) were long (up to 0.9 pm), straight and showed no branching except at the ends of the aggregates, where they were continuous with narrower, tortuous, branching tubules of sarcoplasmic reticulum (Plates 6 and 8). The aggregates were composed of up to 65 tubules. The tubular walls consisted of single, trilaminar membranes about 65 A in thickness. In cross sections the tubules showed patterns of arrangement which, as described below, varied from irregular arrays to regular hexagonal lattices. Areas of continuity between some adjacent tubules were observed at the ends of the aggregates (Plate 8). The lumina of the tubules contained small amounts of flocculent material of moderate electron density (Plates 5, 6, 8, 10 and 1 I). This material appeared condensed against the inner aspects of the tubular walIs (Plate El), and such condensations often formed small spicules that extended into the luminal spaces (Plates 5, 6, 8, 10 and 11). Abnormal Z band material of the type described above was present between the tubules in many of the aggregates (Plates lto3,5,6,8toll).The65Athickfil aments in this material were parallel to the tubules and were not attached to the tubular walls. The electron-dense Z band material associated with the aggregates of tubules often showed a square lattice type of substructure (Plates 9 and 10) which was similar to that in the abnormal Z bands described previously (Plate 5). The electron-dense Z band material in the intertubuIar spaces often appeared continuous with that of some of the adjacent, abnormal Z bands (Plates 9 and 10). The amount of this material that was associated with the aggregates varied according to the size and stage of organization of each aggregate (Plates 9 to 11). Tubules in small (10 tubules or less) aggregates were widely spaced, varied greatly in diameter and appeared irregularly shaped when viewed in cross-section (Plate 9). These small aggregates were associated with the largest masses of abnormal Z band material of the type described above.
AGGREGATES
OF
TUBULES
253
In aggregates showing a more regular pattern of organization the tubules were more cylindrical, more regular in diameter, and more closely packed together (Plate 10). Small amounts of electron-dense Z band material and associated 65 A thick filaments were often present in intertubular spaces of these aggregates. These two components formed partially discontinuous layers that surrounded individual tubules. These layers usually were single; occasionally, two to four parallel layers were present and showed a periodic structure resembling that of the abnormal Z band materiai described above. As in the smaller aggregates discussed above, the filaments between tubules extended beyond the aggregates. In the largest, most highly organized aggregates, the tubules were cylindrical, closely spaced, arranged in a highly regular, hexagonal pattern, and were associated with little or no Z band material (Plate 11). Only single layers, composed of 65 A filaments, with or without associated electron-dense material, were present between these tubules. The center-to-center distance between tubules in these hexagonal lattices varied from 660 to 830 A in patient No. 1 and from 900 to 1200 A in patient No. 2. The diameter of the individual tubules measured from 500 to 875 A and from 560 to 1130 A in these patients, respectively. The second type of aggregates of tubules was observed in cardiac muscle cells exhibiting proliferation and dilatation of sarcoplasmic reticulum and increased numbers of glycogen particles, but intact myofibrils (Plates 12 and 13). The tubules in these aggregates (Plates 12 and 13) were straight, long, unbranched, irregularly arranged, and located in perinuclear areas of cytoplasm. They ranged from 500 to 830 A in diameter and their walls consisted of single, trilaminar membranes measuring 65 A in thickness. These tubules were continuous with those of the sarcoplasmic reticulum, had clear lumina, and were not associated with intertubular filaments or with normal or abnormal Z band material.
4. Discussion
Numerous reports have documented the presence of aggregates of tubules in normal and abnormal striated muscle, but no attempts have been made to classify the various types of aggregates thus far described. Because clarification of the relationships between these aggregates is a prerequisite for discussing the significance of the findings presented in this communication, we propose the system of classification outlined in Tables 2, 3 and 4, in which aggregated tubules in muscle tissue are divided into morphologically disti.nct categories. The most basic distinction between these various types of aggregated tubules concerns whether or not they are composed of single membranes (single-walled tubules, Tables 2 and 3) or of two concentric, separate membranes (double-walled tubules, Table 4). Aggregates of single-walled tubules were subclassified according to whether or not the tubules showed branching within the aggregates (Tables 2
Vastus
Vastus
Schutta & Armitage [59] Miledi & Slater [4Z]
medialis
medialis
Diaphragm
Deltoid
Engel & Dale [21]
Tibialis anterior; gastrocnemius Retropharyngeal rhabdomyoma Breast
Muscle
Snecies
&fi) (1 Pt) Rat
Condition or disease state
branching
described
200
2407
350425
520-580
270-320
300-500
430
300
Overall diameter of tubules (4
tubules
In tissue culture Hyperkalemic periodic paralysis In tissue culture Autophagic glycogenosis$ Hypokalemic periodic paralysis Denervation
Rhabdomyoma
Polymyositis
of single-walled,
Man (1 pt) Man (1 pt) 1 I day old chick embryos Man (1 Pt) 11 day old chick embryos Man
data on aggregates
Breast
etal. [48]
2. Morphologic
Cornog & Gonatas 1141 Ezerman & Ishikawa [23] MacDonald et al. [491 Ishikawa [34]
Palmeiro
Authors (Ref. No.)
TABLE
490
425t
440t
630-730
830-970
220-325
.--
m*
Intertubular distance
in muscle
Hexagonal
Honeycomb
Hexagonal
Hexagonal:
Hexagonal
Irregular
Hexagonal
Hexagonal
Tubular arrangement
tissues -
Fibrillar material EDM
Clear
Fibrillar material Clear
Fibrillar material Flecks of EDMt Clear
Content tubular
of
SR
T
T
T
-
T
T
SR
--._
Tubular connections with
All plates are electron patient No. 2 and Plates
micrographs of degenerated 12 and 13 are from patient
cardiac No. 3.
muscle
PLATE 1. Low power electron microgwph of part of a degenerated an aggregate of hexagonally arranged tubules surrounded by larger tubules of sarcoplasmic reticulum. Myofibriis are absent. x 20 100. PLATE aggregates
2. of reticulum and arrays of thin
cells.
Plates
cardiac branching
1 to 11 are from
muscle cell showing and anastomosing
Low power view of part of a degenerated cardiac muscle cell showing six individual tubules (arrowheads) in various stages of development: dilatation of sarcoplasmic loss of normal myofibrillar structure. Masses of Z band material and disorganized (65 .& diameter) filaments are scattered throughout the celi. x 18 000.
PLATE 3. Part of the same cardiac muscle cell shown in Plate 2. Two aggregates of tubules neat the center are surrounded by several bundles of thin myofilaments attached to masses of 2, band material, Thick myofilamcots are absent. x 30 750. PLATE 4. High power view of a mass of Z band material. The course through the Z band material show a 150 to 200 A periodicity axes. x 78 750.
65 A thick parallel
myofilaments which to their longitudinal
PLATE 5. Three masses of electron-dense Z band material, closely associated with aggregate of tubules, are shown in cross section. The abnormal masses of 2 band material strate a quare lattice type of substructure. This substructure is also evident in the Z band associated with thin (65 A diameter) filaments in the intertubular spaces. x 78 000. PLATE 6. An aggregate of irregularly narrow tubules of sarcoplasmic reticulum intertubular spaces. x 64 500.
shaped tubules shows continuity (arrowheads). A mass of 2 band
a small demonmater&I
with adjacent, more material occupies the
PLATE 7. Many small accumulations of abnormal 2 band material are associated with tubules of sarcoplasmic reticulum. Disorganized myofilaments, 65 A in diameter, are attached masses of Z band material. x 30 000.
dilated to the
PLATE 8. Aggregates of tubules, viewed in longitudinal section, are connected with the sarcoplasmic reticulum (large arrowheads). Continuity between adjacent tubules is seen at the ends of the aggregates (small arrowheads). Thin filaments (65 A in diameter: are present between the tub&s. Small flecks of electron dense material are seen within the aggregated tubules and also wit&r the adjacent tubules of sarcoplasmic reticulum. X 61 500. PLATES
9 to 11, Three
stages in the morphogenesis
PLATE 9. Several accumulations structure are closely associated with x 63 000.
of Z band material irregularly arranged;
PLATE 10. Tubules with a more regular similar in structure to that shown in Plate x 68250. PLATE filaments.
of aggregated which dilated
tubules.
show a square lattice tubules of sarcoplasmic
pattern of arrangement. 9, is continuous with
A mass of Z band material intertubular Z band material.
1 I. Aggregate of highfy organized, hexagonally arranged tubules No abnormal Z band material was present surrounding this aggregate.
PLATE 12. Aggregate of irregularly arranged, straight, unbranched tubules to the nucleus. Most tubules are seen in longitudinal section. x 51 000. PLATE :< 51 600.
13. Aggregate
of irregularly
arranged
tubules,
most
type of subreticulum.
of which
with intertubular x 82 500. is located
are seen in cross
adjacent section.
-_ .--
PLATE 8
et al.
adjacent
Rat
Man;
Desert
t Determination
Polymyositis; Hyperthyroidism Denervation Atrophy
Normal
Coxsackie myocarditis In tissue culture Polymyositis
Myotonic dystrophy Polymyositis
SR = sarcoplasmic reticulum.
tubules;
(1 pt) (1 pt)
iguana
Man (7 pts) Man (4 pts) Man (1 pt) 19 day old fetal mouse Man (3 pts)
Pt = patient; T = T-tubule; reticulum; ER = endoplasmic
between
Heart (endocardial endothelium) Heart (capillary endothelium) Soleus Gastrocnemius
Skeletal
Skeletal (capillary endothelium) Heart (capillary endothelium) Hindlimb
Gastrocnemius
* Center-to-center distance = No data available.
& [SO]
[26]
[51]
etnl.
etal.
ABBREVIATIONS:
-
[49]
et al. [30]
etal.
Pellegrino Franzini
Ferrans
Pham
Gyorkey
Pappas
[31]
[44]
[58]
Haas & Yunis
Norton
Schotland
-
-
-
-
-
-
reticulum;
EDM
Irregular network Hexagonal
Irregular network Rope-like
Irregular network Irregular network Hexagonal
Hexagonal
Clear
EDM
Clear
EDM
Clear
EDM
Clear
-
RER
T
details;
SR
ER
RER
T
ERt
RER
T
endoplasmic
for further
(in some)
= rough
z See reference dense material;
illustrations; = electron
by us from published
250t
1000
1000-l
300-350
250-750
200-220
450t
230
200-250
260-390
Heart
(2 pts)
Man
Bat Cardiac hypertrophy (AI, ASH)
Normal
ABBREVIATIONS:
Pt = patient; T = T-tubule; reticulum; ER = endoplasmic
tissues
570t
430t
500-l
130
600-700
500-830
660-1200
800t
lOOOf
-
100-230 700-1400
1000
Irregular Irregular
6407
590-960
Hexagonal; irregular
Hexagonal
Irregular
Hexagonal
Hexagonal
Irregular
1140t
Hexagonal Irregular
Tubular arrangement
in muscle
Intertubular distance* (4
described
1050t 1500-2000
Overall diameter of tubules (4
tubules
SR = sarcoplasmic reticulum; EDM reticulum; AI = aortic insufficiency;
$ Patients
shown
Amorphous material Spicules and homogeneous EDM
Clear
Granular
Clear
Clear Dense particles (1400 A diameter)
Flecks of EDM
Clear Granular
Content of tubular lumen
= electron dense material; RER = rough ASH = asymmetric septal hypertrophy.
by us from published illustrations; - = No data available. 4).
Local injection of botulinum toxin Cardiac hypertrophy (AI)
Cold injury Familial periodic paralysis Familial hypokalemic periodic paralysis Polymyositis Hypokalemic periodic paralysis with hyperthyroidism Myasthenia gravis
Condition or disease state
non-branching
t Determined * Center-to-center distance between adjacent tubules; different types of aggregated tubules in the same muscle cells (see Table
Present study
With associatedjilaments Cricothyroid Cho et al. [II]
Cardiac
Gastrocnemius Man (1 Pt)
[18]
Duchen
Present study
Man (1 Pt) Mouse
et al. [5]
Bergman
Gastrocnemius
Man (1 pt) Man (2 pts)$
et al. [45]
Rat Man (1 Pt): Man (1 Pt)
Species
of single-walled,
Deltoid Chou [12,13] Bergman et al. [4] Quadriceps and Dunkle et al. [19] femoris
Odor
Muscle
data on aggregates
associated filaments Tibialis anterior Quadriceps femoris Vastus lateralis
3. Morphologic
Without Price et al. [55] Gruner [29]
Authors (Ref. No.)
TABLE
two endoplasmic
to have
SR
T and SR
SR
-
-
-
SR
SR SR
Tubular connections with
et al. [52]
[29]
4. Morpholigic
Vastus medialis (Type II fibers)
Lewis et al. [39]
Man (I pt)
Man (2 PWS
Man (24pts)
Man (1 pt)S Mouse
Specres
tissues
-
6OOt
950t
6OOt
-
of Intertubular distance* (4
in muscle
Diameter tubules (outer/inner) (4
described
Condition or disease state
tubules
Familial periodic 450-500/ 150 paralysis Implantation 500-650/200-300 of benzpyrene Hypokalemic and 700/430 hyperkalemic periodic paralysis and a variety of other conditions$ Hypokalemic 525/300 periodic paralysis with hyperthyroidism 800/400 Presumed acute muscle infarction
of double-walled
Irregular
Hexagonal Irregular
Hexagonal
Hexagonal
Hexagonal
Tubular arrangement
Dense/clear
Clear/clear
Dense flecks of EDM Clear/dense
Clear/clear
Content of tubules (outer/inner)
-II
SR (lateral sacs)
ERTl
SR
Tubular connections with
ABBREVIATIONS:
Pt = patient; reticulum; -
T = T-tubule; SR = = No data available.
sarcoplasmic
reticulum;
EDM
=
electron
dense
material;
ER
=
endoplasmic
* Center-to-center distance between adjacent tubules; $ Patients shown to have two t Determination by us from published illustrations; different types of aggregated tubules in the same muscle cells (see Table 3) ; 5 See reference for further 7 These tubules were interpreted details; as being virus-like particles derived from endoplasmic reticulum; /I These tubules were interpreted as possibly representing abnormal mitochondria.
Quadriceps femoris
Lumbosacral (Macrophages) Skeletal (Type II fibers)
Quadriceps
Muscle
data on aggregates
Bergman et al. [4] and Dunkle etal. [19]
Engel et al. [.?2]
Popoff
Gruner
Authors (Ref. No.)
TABLE
258
B. J. MARON
AND
V. J. FERRANS
and 3). Aggregates of branching, single-walled tubules (Table 2) were further identified depending on whether their components were continuous with the T system [14, 21, 23, 34, 49, 50, 58, 591 or with the sarcoplasmic reticulum [26, 39, 31, 41, 44, 48, 511. Most aggregates of non-branching, single-walled tubules (Table 3) were reported as being derived from components of sarcoplasmic reticulum [29, 45, 551; in one report [II] these aggregates were described as continuous with both the sarcoplasmic reticuIum and the T system. The two types of aggregates described in the present communication belong to this category of non-branching, single-walled tubules derived from the sarcoplasmic reticulum. This constitutes the first description of such structures in cardiac muscle. Aggregates of double-walled tubules (Table 4) have been described in skeletal muscle [4, 19, 22, 29, 39, 521, but not in cardiac muscle; they bear little resemblance to other types of aggregates and are of uncertain origin. Aggregates of single-walled, branching tubules derived from the T system have been described as arranged either irregularly [23] or in highly regular, lattice-like configurations [la, 21, 34, 49, 50, 58, 591, and have been demonstrated to arise from inpocketings of the plasma membranes [34]. Definition of the relation between aggregates of branching tubules and the transverse tubular system has been achieved by demonstrating the passage of ferritin from the extracellular space and T tubules into the lumina of tubules in the aggregates [23, 341. Aggregates of single-walled, branching tubules derived from endoplasmic reticulum have been described most frequently in a number of conditions which are classified as autoimmune diseases, particularly systemic lupus erythematosus [see 30, 57, 60 for reviews] as we11 as in numerous viral infections [see 57 and 60 for reviews]. These aggregates have been studied most extensively in glomerular endothelium. They have not been described in cardiac muscle cells, but have been reported in capillary endothelium of the heart [26, 311. Similar aggregates have been reported in skeletal muscle cells [30] and capillary endothelium [44] of patients with polymyositis. These aggregated tubules have small diameters (200 to 300 A) and short lengths, and are commonly located within cisterns of rough endoplasmic reticulum, but also occur between the inner and the outer nuclear membranes [26] and even within nuclei [IO, 28, 36, 431. They form complex threedimensional networks of tubules, the ultrastructural appearance of which depends on the plane of section [9]. Their pattern of branching varies considerably, and in a number of reports the branching tubules have been shown to form highly regular crystalline aggregates [2, 7, 8, 17, 40, 48, 561. These branching tubules differ clearly from those found in the 3 patients reported in this communication. Although aggregates of branching tubules have been considered by some to have a structural similarity to nucleocapsids of viruses of the myxo- or paramyxovirus group [30], other investigators have shown that myxoviruses and these tubules differ morphologically [GO] and histochemically [57]. It is most likely that aggregates of this type represent a nonspecific response of the endoplasmic reticulum to cellular injury or
AGGREGATES
OF TUBULES
259
viral infection. The characteristic locations of the branching tubules just described serve to differentiate them from proliferating tubules of endoplasmic reticulum which occur free in the cytoplasm and do not become aggregated into discrete masses. The latter type of tubular proliferation can be induced in liver cells by a variety of drugs [16’, 20, 33, 46, 541. It has been our experience that proliferation of sarcoplasmic reticulum, without aggregation of tubules, is a common finding in hypertrophied cardiac muscle cells. Aggregates of long, straight, single-walled tubules were subdivided into 2 categories according to whether or not intertubular filaments were present within the aggregates (Table 2). Aggregates of single-walled tubules without associated filaments have been described in human skeletal muscle in familial [29] and hypokalemic [4, 19, 451 types of periodic paralysis, myasthenia gravis [IS], polymyositis, 112, 131 and in experimental skeletal muscle injury [18, 551. Aggregates of long, straight and unbranched tubules in all of these described studies and in this communication were found only in muscle cells showing evidence of degeneration or atrophy. The aggregates described by Chou [12, 133 clearly differ from all the others in the category of single-walled, straight tubules in that they were very small in diameter (100 to 230 A), were present within nuclei as well as in the cytoplasm, and were considered to be myxovirus-like. With this exception the aggregates of single-walled, straight tubules without associated filaments (Table 3) are morphologically similar to those described in patient No. 3 in this report. These aggregates differed from those found in our patients No. 1 and No. 2 in that they were not associated with Z band material or intertubular filaments and did not show a highly ordered arrangement. Aggregates of tubules found in cardiac muscle cells of patient No. 3 and in skeletal muscle cells of patients in other reports (Table 3) differ morphologically from crystalline aggregates of microtubules [3J. The latter aggregates have been induced in vitro by periwinkle alkaloids in L-strain fibroblasts and in human leukocytes [3], but have not been described in muscle cells. The microtubules in such aggregates appear cylindrical and are of much smaller diameter (270 A) than the straight-walled tubules in striated muscle. The latter tubules also differ morphologically from annulate lamellae, which are a component of many embryonic, germ or tumor cells and consist of parallel paired membranes (300 to 500 A apart), interrupted at regular intervals by pores [371. Our interpretation of the nature of the tubules in patient No. 3 are that they are greatly elongated, moderately dilated components of sarcoplasmic reticulum. We conclude that these tubules represent an unusual type of proliferation of sarcoplasmic reticulum in cardiac muscle cells that have normally arranged contractile elements. The aggregatesof single-walled tubules associatedwith intertubular filamentous material, which we observed in degenerated cardiac muscle cells from patients No. 1 and No. 2, resemblethosedescribed in cricothyroid muscleof bats [II]. The latter is the only report of aggregated tubules in normal muscle cells. The aggregated tubules in bat skeletal muscle differed significantly from those in our patients in
260
B. J. MARON
AND
V. J. FERRANS
that they had connections with both the sarcoplasmic reticulum and the T system. Furthermore, these tubules and their intertubular filaments were not associated with either normal or abnormal Z band material. Ribosomes are the only other type of structure reported to occur in intertubular spaces of aggregated tubules. Such aggregates, which have been described only in chief cells of bat gastric mucosa [35], were arranged in hexagonal lattices and were composed of long, straight tubules of rough-surfaced endoplasmic reticulum. The tubules found in myocardium of patients No. 1 and No. 2 clearly differ from several types of double-walled tubules that have been reported principally in human skeletal muscle myopathies (Table 4). The exact origin of these double-walled tubules is unclear. According to Engel et al. [.?,?I th ese tubules are connected to the sarcoplasmic reticulum and arise from proliferation of its components. Lewis et al. [39] believe, however, that they are derived from mitochondria. Our concept of the morphogenesis of the hexagonally arranged aggregates of tubules found in two of the patients (Nos. 1 and 2) in this report is illustrated in Plates 9 to 11. Initially, marked focal proliferation and dilatation of the sarcoplasmic reticulum occurs in association with large masses of abnormal Z band material to which thin (actin) filaments are attached. In the early stages of formation of the aggregates, the tubules of sarcoplasmic reticulum appear as irregularly shaped cylinders surrounded by Z band material and actin filaments, and the distance between adjacent tubules is not uniform. These tubules appear to be precursors of the hexagonally arranged tubules in larger aggregates which are not associated with Z band material. As the mass of Z band material surrounding the tubules disappears, the tubules become more cylindrical, more closely associated and evenly spaced. The intertubular filaments and associated electron-dense material probably represent portions of the actin filaments and masses of Z band material previously present in the intertubular spaces. A close relationship was evident between developing aggregates of tubules and abnormal myofibrils composed of irregularly-shaped masses of Z band material and associated thin myofilaments. The masses of abnormal Z band material observed in patients Nos 1 and 2 resemble those described by Munnell and Getty [42] in ventricular myocardium from old dogs, by C&C et al. [15] in atria1 muscle from patients with rheumatic heart disease and by Legato [38] in atria1 and ventricular muscle from patients with a variety of congenital cardiac lesions and in embryonic rat ventricular muscle grown in tissue culture. In all these instances the Z band material showed a periodic arrangement due to the presence of filaments that were spaced 200 A apart and were oriented parallel to the longitudinal axis of the myofibrils. Such an arrangement of Z band material differs from that described by Fawcett [24] in myocardium from old cats and by Bishop and Cole [S] in hypertrophied and failing canine myocardium in that it lacks the well defined transverse (i.e., perpendicular to the longitudinal axes of the myofibrils) periodicity of about 200 A found in the Z band material observed by these authors. The significance of
AGGREGATES
OF TUBULES
261
these differences in arrangement of Z band material is unclear. Legato has postulated [38] that focal accumulations of Z band material represent early stages in the formation of new sarcomeres during normal growth or hypertrophy of cardiac muscle cells. We believe that the masses of Z band material in abnormal myofibrils which lacked thick myofilaments are indicative of degeneration rather than of hypertrophy. This degeneration was characterized by selective loss of thick (myosin) myofilaments from the cardiac muscle cells and was present in variable degrees of severity in biopsies from 13 of the 24 patients in this study. Only in 2 of these 13 patients, however, did we find aggregates of tubular structures associated with intertubular material. These observations are consistent with the hypothesis that such aggregates of tubules form only in those cardiac muscle cells in which the sarcoplasmic reticulum is still capable of proliferating after this type of myofibrillar degeneration develops. It is not known whether Z bands, T tubules and thick myofilaments play a direct role in inducing the normal morphogenesis of sarcoplasmic reticulum components which either surround the myofibrils (longitudinal elements) or are closely associated with Z bands and T tubules (terminal cisterns). The observations in this report suggest that in the presence of advanced degenerative changes (i.e., abnormal masses of Z band material and absence of T tubules and thick myofilaments) growth of the sarcoplasmic reticulum proceeds in a highly abnormal fashion. Data from other studies suggest that this is also the case in skeletal muscle cells, as evidenced by reports of the presence of isolated masses of thin myofilaments in cells containing aggregates of tubules [.5, 4.51. Thus, we conclude that the formation of aggregates of tubules derived from the sarcoplasmic reticulum is a degenerative change related to the inability of the sarcoplasmic reticulum to form meshworks around abnormally constituted myofibrils, or terminal cisterns around T tubules. REFERENCES 1. 2. 3. 4. 5. 6.
ANDERSON-CEDERGREN, E. Ultrastructure of motor end plate and sarcoplasmic components of mouse skeletal muscle fiber. Journal of Ultrastructure Research, Supplement 1, pp. 126-166 (1959). BARINGER, J. R. & GRIFFITH, J. F. Experimental herpes encephalitis: Crystalline arrays in endoplasmic reticulum. Science 165, 1381 (1969). BENSCH, K. G. & MALAWISTA, S. E. Microtubular crystals in mammalian cells. JournaE of Cell Biolou 40, 95-107 (1969). BERGMAN, R. A., AFIFI, A. K., DUNKLE, L. M. & JOHNS, R. J. Muscle pathology in hypokalemic periodic paralysis with hyperthyroidism. II. A light and electron microscopic study. The Johns Hopkins Medical Journal 126, 100-l 17 (1970). BERGMAN, R. A. & JOHNS, R. J. Ultrastructural alterations in muscle from patients with myasthenia gravis and Eaton-Lambert syndrome. Annals of the .New York Academy of Sciences 183, 88-122 (1971). BISHOP, S. P. & COLE, C. R. Ultrastructural changes in the canine myocardium with right ventricular hypertrophy and congestive heart failure. Laboratory Investigation 20, 219-229 (1969).
262
B. J. MARON
AND
V. J. FERRANS
7. BLOODWORTH, J. M. B. & SHELP, W. D. Endothelial cytoplasmic inclusions. Archives of Pathology 90, 252-258 (1970). 8. CHANDRA, S., KUZMIC, S., BRANDT, P. M. & MONROE, J. H. Microtubular formations of endoplasmic reticulum in human pathological tissues. Proceedings of the Twentyjfth Annual Meeting, Electron Microscojy Society of America (C. J. Plrceneaux, Ed.) pp. 172173. Chicago, Illinois : Claitor’s Book Store ( 1967). 9. CHANDRA, S. Undulating tubules associated with endoplasmic reticulum in pathologic tissues. Laboratory Investigation 18, 422-428 (1968). 10. CHANDRA, S., BROWN, D. E., ALDENDERPER, P., GARON, C., BUSCHECK, F. T. & MANAKER, R. A. Morphologic and serologic studies of transplanted human leukocyte culture (M-l) cells in laboratory animals. Cancer Research 29, 1829-1839 (1969). 11. CHO, Y., SIDIES, J. M. & DE BRWN, P. P. H. Electron microscopic studies on a tubulofilamentous fasciculus in the bat cricothyroid muscle. Journal of Ultrastructure Research 41, 344-357 (1972). structures in a case of human chronic polymyositis. 12. CHOU, S-M. Myxovirus-like Science 158, 1453-1455 (1967). structures and accompanying nuclear changes in chronic 13. THOU, S-M. Myxovirus-like polymyositis. Archives of Pathology 86, 649-658 ( 1968). of rhabdomyoma. 3ournal of 14. CORNOG, J. L., JR. & GONATAS, N. K. Ultrastructure 15. 16. 17. 18. 19. 20. 21.
22. 23. 24. 25.
Ultrastructure Research 20, 433-450 (1967). CC~T~, G., MOHIUDDIN, S. M. & ROY, P. E. Occurrence of Z-band widening in human atria1 cells. Exfierimental and Molecular Pathology 13, 307-318 (1970). DALLNER, G., BERGSTRAND, A. & NILSSON, R. Heterogeneity of rough-surfaced liver microsomal membranes of adult phenobarbital-treated, and newborn rats. Journal of Cell Biolou 38, 257-276 (1968). DE MARTINO, C., ACCINI, L. & ANDRES, G. A. Tubular structures associated with the
endothelial endoplasmic reticulum in glomenrlar capillaries of rhesus monkey and nephritic man.
AGGREGATES ROBERTS, W. C. Cardiac Preservation: Studies on Cardiac Structure and Metabolism,
26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
OF TUBULES
263
A morphologic study. In Recent Advances in Volume 1, Myocardiology (E. Bajusz and G. Rona, Eds.) pp. 351-375. Baltimore: University Park Press. FERRANS, V. J., MASSUMI, R. A., [SHUGOLL, G. E., ALI, N. & ROBERTS, W. C. Ultrastructural studies of myocardial biopsies in 45 patients with obstructive or congestive cardiomyopathy. In Recent Advances in Studies on Cardiac Structure and Metabolism. Volume 2, Cardiomyopathies. (E. Bajusz, G. Rona, A. J. Brink, A. Lochner, Eds) Baltimore: University Park Press, pp. 231-272 (1973). FREEMAN, J. & SPURLOCK, B. A new epoxy embedment for electron microscopy. Journal of Cell BioloQ 13, 437-443 (1962). GIVAN, K. F. & JEZEQUEL, A.-M. Infectious canine hepatitis: a virologic and ultrastructural study. Laboratory Investigation 21, 36-45 (1969). GRUNER, J.-E. Anomalies du reticulum sarcoplasmique et proliferation de tubules dans le muscle dune paralysie periodique familiale. Corn&es Rendus des Sbances de la Societe’ de Biologie, Paris 160, 193-195 (1966). GYORKEY, F., SINKOVICS, J. G., MIN, K. W. & GYORKEY, P. A morphologic study on the occurrence and distribution of structures resembling viral nucleocapsids in collagen diseases. American Journal of Medicine 53, 148-158 (1972). HAAS, J. E. & YUNIS, E. J. Viral crystalline arrays in human Coxsackie myocarditis. Laboratory Investigation 23, 4424% (1970). HIBBS, R. G., FERRANS, V. J., BLACK, W. C., WEILBAECHER, D. G., WALSH, J. J. & BURGH, G. E. Alcoholic cardiomyopathy: An electron microscopic study. American Heart 3ournal69, 766-779 (1965). H~I-TERER, F., SCHAFFNER, F., KLION, F. M. & POPPER, H. Hypertrophic, hypoactive smooth endoplasmic reticulum: A sensitive indicator of hepatotoxicity exemplified by dieldrin. Science 161, 1017-1019 (1968). ISHIKAWA, H. Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. Journal of Cell Biology 38, 5166 (1968). ITO, S. & WINCHESTER, R. J. The fine structure of the gastric mucosa in the bat. Journal of Cell Biology 16, 541-577 (1963). JENSON, A. B., SPJUT, H. J., SMITH, M. N. & RAPP, F. Intracellular branched tubular structures in osteosarcoma. Cancer 27, 1440-1448 (1971). KESSEL, R. G. Annulate lamellae. Journal of Ultrastructure Research, Supplement 10, l-82 (1968). LEGATO, M. J. Sarcomerogenesis in human myocardium. Journal of Molecular and Cellular Cardiology 1, 425-437 (1970). LEWIS, P. D., PALLIS, C. & PEARSE, A. G. E. “Myopathy” with tubular aggregates. Journal of the jyeurological Sciences 13, 381-388 (1971). MACDONALD, R. D., REWCASTLE, N. B. & HUMPHREY. J. G. The myopathy of hyperkalemic periodic paralysis. An electron microscopic study. Archives of .Neurolopy 19, 274-283 (1968). MILEDI, R. & SLATER, C. R. Electron-microscopic structure of denervated skeletal muscle. Proceedings of the Royal Society of London, Series B, 174, 253-269 (1969). MUNNELL, J. F. & GE~Y, R. Canine myocardial Z-disc alterations resembling those of Nemaline myopathy. Laboratory Investigation 19, 303-308 (1968). NIELAND, N. W., HASHIMOTO, K. & M&I, A. T. Microtubular inclusions in normal skin of systemic lupus erythematosus patients. Arthritis and Rheumatism 15, 193-200 (1972). NORTON, W. L., EDWARD, V. & ROBISON, L. Endotheiial inclusions in dermatomyositis. Annals of Rheumatic Diseases 29, 67-72 (1970).
264 45.
46.
47. 48. 49.
50. 51.
52.
53.
54. 55.
56. 57.
58. 59. 60.
B. J. MARON
AND
V. J. FERRANS
ODOR, D. L., PATEL, A. N. & PEARCE, L. A. Familial hypokalemic periodic paralysis with permanent myopathy. A clinical and ultrastructural study. Journal of Neuropathology and Experimental Neurology 26, 98-114 (1967). ORRENIUS, S. & ERICSSON, J. L. E. On the relationship of liver glucose-6-phosphatase to the proliferation of endoplasmic reticulum in phenobarbital induction. Journal of Cell Biology 31, 243-256 (1966). PAGE, E. & MCCALLISTER, L. P. Quantitative electron microscopic description of heart muscle cells. American Journal of Cardiology 31, 172-181 (1973). PALMEIRO, J., BEHREND, R. C. H. & WECHSLER, W. Elektronenmikroskopische Befunde an der Skeletmuskulatur bei Polymyositis. Acta ~uropathologi&a 7, 26-43 (1966). PAPPAS, G. D., PETERSON, E. R., MASUROVSKY, E. B. & GRAIN, S. M. Electron microscopy of the in vitro development of mammalian motor end plates. Annals of the New York Academy of Sciences 83, 33-45 (1971). PELLEGRINO, C. & FRANZINI, C. An electron microscope study of denervation atrophy in red and white skeletal muscle fibers. Journal of Cell Biology 17,327-349 (1963). PHAM, T. D., LUSE, S. A. & DEMPSEY, E. W. A unique form of endoplasmic reticulum in endocardial endothelia of the desert iguana. Journal of Ultrastructure Research 39, 149-162 (1972). POPOFF, N., SUTTON, C. H. & ZIMMERMAN, H. M. Virus-like particles in reactive cells associated with crystals of implanted carcinogen. Acta Neuropathologica 10, 308-323 (1968). PORTER, K. R. & PALADE, G. E. Studies on the endoplasmic reticulum. III. Its form and distribution in striated muscle cells. Journal of Biophysical and Biochemical Cytolopv 3, 269-301 (1957). POSALAKI, Z. & BARKA, T. Alterations of hepatic endoplasmic reticulum in porphyric rats. Journal of Histochemistry and Cytochemistry 16, 337-345 (1968). PRICE, H. M., Howns, E. L. & BLUMBERG, J. M. Ultrastructural alterations in skeletal muscle fibers injured by cold. I. The acute degenerative changes. Laboratory Investigation 13, 1264-1278 (1964). ROSEN, S. & TISHER, C. C. Observations on the rhesus monkey glomerulus and juxtaglomerular apparatus. Laboratory Investigation 18,240-248 (1968). SCHAFT, Z., HEINE, U. & DALTON, A. J. Ultramorphological and ultracytochemical studies on tubuloreticular structures in lymphoid cells. Cancer Research 32, 2696-2706 (1972). SCHOTLAND, D. L. An electron microscopic investigation of myotonic dystrophy. Journal of Neuropathology and Experimental NewolD 29, 241-253 (1970). SCHUTTA, H. S. & ARMITAGE, J. L. Thyrotoxic hypokalemic periodic paralysis. A fine structure study. Journal of Neuropathology and Experimental .Neurolopy 28, 321-336 (1969). UZMAN, B. G., SAITO, H. & KASAC, M. Tubular arrays in the endoplasmic reticulum in human tumor cells. Laboratory Investigation 24, 4922498 (1971). Note added in proof: After this manuscript was accepted for publication, we have observed aggregates of tubules, of the type shown in Figures l-l 1, in ventricular septal myocardium from a 10 year-old boy with discrete membranous subaortic stenosis and asymmetric septal hypertrophy.
of Molecular
and Cellular
Aggregates
Cardiology
(19741,
of Tubules
in Human
BARRY J. MARON Cardiology
Branch
6,249-264
AND
VICTOR
and the Section of Pathology, .National of Health, Bethesda, Maryland (Received
30 July
1973,
Cardiac
Muscle
Cells
J. FERRANS
Heart and Lung Institute, 20014, U.S.A.
and accepted 4 October
.National Institutes
1973)
B. J. MARON AND V. J. FERRANS. Aggregates of Tubules in Human Cardiac Muscle Cells. Journal of Molecular andCellular Cardiology (1974) 6,249-264. Two types ofaggregated tubules derived from, and continuous with, elements of sarcoplasmic reticulum were observed in degenerated cardiac muscle cells from patients with cardiac hypertrophy. Aggregates of the first type, found in 2 patients, were composed oflong, parallel tubules which measured from 500 to 1130 A in diameter and showed different degrees of organization. In some of these aggregates the tubules were irregularly arranged and were closely associated with large masses of abnormal 2 band material to which thin myofilaments were attached. In other instances, the tubules formed highly regular, hexagonally arranged aggregates; the spaces between these aggregated tubules contained thin myofilaments and small amounts of Z band material. Cells in which these aggregates were present showed advanced degenerative changes characterized by disarray of myofibrils, absence of T tubules, and selective loss of thick myofilaments. It is concluded that the formation of these aggregates of tubules derived from the sarcoplasmic reticulum is a degenerative change related to the inability of the sarcoplasmic reticulum to form meshworks around abnormally constituted myofibrils, or terminal cisterns around T tubules. Aggregates of the second type, found in one patient, were formed by long, straight, irregularly arranged tubules that measured 500 to 830 A in diameter and were not associated with myofibrillar components. These aggregates are considered to represent an unusual type of proliferation of the sarcoplasmic reticulum in cells having normally arranged contractile elements. KEY
WORDS:
degeneration;
Cardiac Aortic
hypertrophy; regurgitation;
Ultrastructure; Hypertrophic
Sarcoplasmic cardiomyopathy.
reticulum;
Myofibrillar
I. Introduction The sarcoplasmic reticulum of cardiac muscle cells is a specialized network of intracellular tubules which are connected with the outer nuclear membranes and form a meshwork around each myofibril. The sarcoplasmic reticulum has specialized areas, known as terminal cisterns, which are closely approximated to the extracellular environment by virtue of their apposition to the plasma membranes. The areas of apposition occur either near the cell surfaces or, deeper in the cell, in relation to portions of the transverse tubular system and to the Z bands of the myofibrils [I, 531. Because of its important role in cardiac contraction, numerous studies have been made of pathologic alterations in the sarcoplasmic reticulum of cardiac muscle
250
B.
J.
MARON
AND
V.
J.
FERRANS
cells. Changes observed include swelling or dilatation of the tubules, which occurs as a non-specific finding in a variety of conditions [25, 321, and increased amounts of morphologically normal tubules, which have been reported in cardiac hypertrophy [47j. The purpose of this communication is to describe alterations of sarcoplasmic reticulum that have not been previously reported in myocardium. These alterations consist of two different types of aggregates of tubules formed by modified components of sarcoplasmic reticulum in degenerated cardiac muscle cells of patients with cardiac hypertrophy.
2. Materials
and
Methods
Observations described in this communication are based on study of myocardial biopsies obtained at operation from the left ventricular apex in 14 patients with aortic valvular disease and from the left ventricular posterior wall in 10 patients with hypertrophic cardiomyopathy (asymmetric septal hypertrophy) . All tissues were fixed in cold 3% glutaraldehyde in 0.1 M-phosphate buffer, pH 7.2. After washing with several changes of cold 5% sucrose in 0.1 M-phosphate buffer, pH 7.2, the tissues were postflxed with 1 y0 osmium tetroxide in Millonig’s phosphate buffer, pH 7.2, dehydrated with a graded series of ethanols and propylene oxide and embedded in Maraglas [27]. Ultrathin sections were stained with uranyl acetate and lead citrate and viewed with either an RCA EMU-3G or JEOL 1OOB electron microscope.
3. Results The morphologic characteristics of the two types of aggregates described in this study are summarized in Table 1. The first type of aggregated tubules was observed in two patients: patient No. 1 was a 44-year-old man with severe aortic regurgitation; patient No. 2 was a 24-year-old man with non-obstructive asymmetric septal hypertrophy (hypertrophic non-obstructive cardiomyopathy) and pulmonary sarcoidosis. The second type of aggregate was observed in a 46-year-old man (patient No. 3) with aortic regurgitation. Aggregates of either type were not found in the other 21 patients. Aggregates of tubules of the first type occurred only in cardiac muscle cells showing features of severe degeneration (Plates 1 and 2). These features were: loss almost complete absence of myosin filaments; of normal myofibrillar structure; clumps of abnormal Z band material; numerous actin filaments, both free in the sarcoplasm and attached to the abnormal Z bands; markedly increased numbers of tubules of sarcoplasmic reticulum, which were focally dilated; unusually small mitochondria; absent T tubules, and thickened basement membranes. In sections cut parallel to the longitudinal axes of the myofibrils, the abnormal Z band
between
500-875 560-l 130 500-830
Overall tubular diameter (4
characteristics
distance
Hexagonal Hexagonal Irregular
1 2 3
* Center-to-center
Arrangement of tubules
1. Morphologic
Patient No.
TABLE
adjacent
50-65 65 -
of
in cardiac
Diameter intertubular filaments (4
of tubules
tubules.
660-830 900-1200 -
Inter-tubular distance* (4
of aggregates
cells
50-65 65 60
Thickness of tubular wall (4
muscle Size of tubular aggregates Cd
5200 0.7 x 1.2 9000 0.7 x 1.6 8500 1.4 x 2.5 _-_-___~
Maximum length of tubules (4
50 65 35
Maximum no. tubules per aggregate
5 7 4
Maximum no. aggregates per cell
252
B.
J.
MARON
AND
V.
J.
FERRANS
material appeared to be traversed by filaments that were arranged in a regular, parallel fashion, measured 65 A in diameter and were separated from each other by distances of approximately 100 A (Plates 3 and 4). Because of their association with Z band material, these filaments appeared more electron dense in the Z band areas than elsewhere. Such filaments corresponded morphologically to the thin (actin) myofilaments and extended as much as 1.5 pm from the centers of the abnormal Z bands, but were not associated with thick myofilaments (Plates 3 and 4). In sections cut perpendicular to the longitudinal axes of these filaments, the abnormal Z band material appeared finely fibrillar and often exhibited a square lattice type of substructure (Plate 5). Adjacent accumulations of Z band material with attached thin myofilaments were often oriented obliquely or perpendicular to each other, indicating a loss of normal myofibrillar alignment (Plates 2 and 3). In some areas, the thin myofilaments attached to the masses of Z band material were tangled and disorganized (Plates 6 and 7) ; in other areas, the aggregations of thin myofilaments appeared to be associated with little or no Z band material (Plates 1 and 2). The tubules constituting the first type of aggregates (Plates 1 to 3, 5 to 11) were long (up to 0.9 pm), straight and showed no branching except at the ends of the aggregates, where they were continuous with narrower, tortuous, branching tubules of sarcoplasmic reticulum (Plates 6 and 8). The aggregates were composed of up to 65 tubules. The tubular walls consisted of single, trilaminar membranes about 65 A in thickness. In cross sections the tubules showed patterns of arrangement which, as described below, varied from irregular arrays to regular hexagonal lattices. Areas of continuity between some adjacent tubules were observed at the ends of the aggregates (Plate 8). The lumina of the tubules contained small amounts of flocculent material of moderate electron density (Plates 5, 6, 8, 10 and 1 I). This material appeared condensed against the inner aspects of the tubular walIs (Plate El), and such condensations often formed small spicules that extended into the luminal spaces (Plates 5, 6, 8, 10 and 11). Abnormal Z band material of the type described above was present between the tubules in many of the aggregates (Plates lto3,5,6,8toll).The65Athickfil aments in this material were parallel to the tubules and were not attached to the tubular walls. The electron-dense Z band material associated with the aggregates of tubules often showed a square lattice type of substructure (Plates 9 and 10) which was similar to that in the abnormal Z bands described previously (Plate 5). The electron-dense Z band material in the intertubuIar spaces often appeared continuous with that of some of the adjacent, abnormal Z bands (Plates 9 and 10). The amount of this material that was associated with the aggregates varied according to the size and stage of organization of each aggregate (Plates 9 to 11). Tubules in small (10 tubules or less) aggregates were widely spaced, varied greatly in diameter and appeared irregularly shaped when viewed in cross-section (Plate 9). These small aggregates were associated with the largest masses of abnormal Z band material of the type described above.
AGGREGATES
OF
TUBULES
253
In aggregates showing a more regular pattern of organization the tubules were more cylindrical, more regular in diameter, and more closely packed together (Plate 10). Small amounts of electron-dense Z band material and associated 65 A thick filaments were often present in intertubular spaces of these aggregates. These two components formed partially discontinuous layers that surrounded individual tubules. These layers usually were single; occasionally, two to four parallel layers were present and showed a periodic structure resembling that of the abnormal Z band materiai described above. As in the smaller aggregates discussed above, the filaments between tubules extended beyond the aggregates. In the largest, most highly organized aggregates, the tubules were cylindrical, closely spaced, arranged in a highly regular, hexagonal pattern, and were associated with little or no Z band material (Plate 11). Only single layers, composed of 65 A filaments, with or without associated electron-dense material, were present between these tubules. The center-to-center distance between tubules in these hexagonal lattices varied from 660 to 830 A in patient No. 1 and from 900 to 1200 A in patient No. 2. The diameter of the individual tubules measured from 500 to 875 A and from 560 to 1130 A in these patients, respectively. The second type of aggregates of tubules was observed in cardiac muscle cells exhibiting proliferation and dilatation of sarcoplasmic reticulum and increased numbers of glycogen particles, but intact myofibrils (Plates 12 and 13). The tubules in these aggregates (Plates 12 and 13) were straight, long, unbranched, irregularly arranged, and located in perinuclear areas of cytoplasm. They ranged from 500 to 830 A in diameter and their walls consisted of single, trilaminar membranes measuring 65 A in thickness. These tubules were continuous with those of the sarcoplasmic reticulum, had clear lumina, and were not associated with intertubular filaments or with normal or abnormal Z band material.
4. Discussion
Numerous reports have documented the presence of aggregates of tubules in normal and abnormal striated muscle, but no attempts have been made to classify the various types of aggregates thus far described. Because clarification of the relationships between these aggregates is a prerequisite for discussing the significance of the findings presented in this communication, we propose the system of classification outlined in Tables 2, 3 and 4, in which aggregated tubules in muscle tissue are divided into morphologically disti.nct categories. The most basic distinction between these various types of aggregated tubules concerns whether or not they are composed of single membranes (single-walled tubules, Tables 2 and 3) or of two concentric, separate membranes (double-walled tubules, Table 4). Aggregates of single-walled tubules were subclassified according to whether or not the tubules showed branching within the aggregates (Tables 2
Vastus
Vastus
Schutta & Armitage [59] Miledi & Slater [4Z]
medialis
medialis
Diaphragm
Deltoid
Engel & Dale [21]
Tibialis anterior; gastrocnemius Retropharyngeal rhabdomyoma Breast
Muscle
Snecies
&fi) (1 Pt) Rat
Condition or disease state
branching
described
200
2407
350425
520-580
270-320
300-500
430
300
Overall diameter of tubules (4
tubules
In tissue culture Hyperkalemic periodic paralysis In tissue culture Autophagic glycogenosis$ Hypokalemic periodic paralysis Denervation
Rhabdomyoma
Polymyositis
of single-walled,
Man (1 pt) Man (1 pt) 1 I day old chick embryos Man (1 Pt) 11 day old chick embryos Man
data on aggregates
Breast
etal. [48]
2. Morphologic
Cornog & Gonatas 1141 Ezerman & Ishikawa [23] MacDonald et al. [491 Ishikawa [34]
Palmeiro
Authors (Ref. No.)
TABLE
490
425t
440t
630-730
830-970
220-325
.--
m*
Intertubular distance
in muscle
Hexagonal
Honeycomb
Hexagonal
Hexagonal:
Hexagonal
Irregular
Hexagonal
Hexagonal
Tubular arrangement
tissues -
Fibrillar material EDM
Clear
Fibrillar material Clear
Fibrillar material Flecks of EDMt Clear
Content tubular
of
SR
T
T
T
-
T
T
SR
--._
Tubular connections with
All plates are electron patient No. 2 and Plates
micrographs of degenerated 12 and 13 are from patient
cardiac No. 3.
muscle
PLATE 1. Low power electron microgwph of part of a degenerated an aggregate of hexagonally arranged tubules surrounded by larger tubules of sarcoplasmic reticulum. Myofibriis are absent. x 20 100. PLATE aggregates
2. of reticulum and arrays of thin
cells.
Plates
cardiac branching
1 to 11 are from
muscle cell showing and anastomosing
Low power view of part of a degenerated cardiac muscle cell showing six individual tubules (arrowheads) in various stages of development: dilatation of sarcoplasmic loss of normal myofibrillar structure. Masses of Z band material and disorganized (65 .& diameter) filaments are scattered throughout the celi. x 18 000.
PLATE 3. Part of the same cardiac muscle cell shown in Plate 2. Two aggregates of tubules neat the center are surrounded by several bundles of thin myofilaments attached to masses of 2, band material, Thick myofilamcots are absent. x 30 750. PLATE 4. High power view of a mass of Z band material. The course through the Z band material show a 150 to 200 A periodicity axes. x 78 750.
65 A thick parallel
myofilaments which to their longitudinal
PLATE 5. Three masses of electron-dense Z band material, closely associated with aggregate of tubules, are shown in cross section. The abnormal masses of 2 band material strate a quare lattice type of substructure. This substructure is also evident in the Z band associated with thin (65 A diameter) filaments in the intertubular spaces. x 78 000. PLATE 6. An aggregate of irregularly narrow tubules of sarcoplasmic reticulum intertubular spaces. x 64 500.
shaped tubules shows continuity (arrowheads). A mass of 2 band
a small demonmater&I
with adjacent, more material occupies the
PLATE 7. Many small accumulations of abnormal 2 band material are associated with tubules of sarcoplasmic reticulum. Disorganized myofilaments, 65 A in diameter, are attached masses of Z band material. x 30 000.
dilated to the
PLATE 8. Aggregates of tubules, viewed in longitudinal section, are connected with the sarcoplasmic reticulum (large arrowheads). Continuity between adjacent tubules is seen at the ends of the aggregates (small arrowheads). Thin filaments (65 A in diameter: are present between the tub&s. Small flecks of electron dense material are seen within the aggregated tubules and also wit&r the adjacent tubules of sarcoplasmic reticulum. X 61 500. PLATES
9 to 11, Three
stages in the morphogenesis
PLATE 9. Several accumulations structure are closely associated with x 63 000.
of Z band material irregularly arranged;
PLATE 10. Tubules with a more regular similar in structure to that shown in Plate x 68250. PLATE filaments.
of aggregated which dilated
tubules.
show a square lattice tubules of sarcoplasmic
pattern of arrangement. 9, is continuous with
A mass of Z band material intertubular Z band material.
1 I. Aggregate of highfy organized, hexagonally arranged tubules No abnormal Z band material was present surrounding this aggregate.
PLATE 12. Aggregate of irregularly arranged, straight, unbranched tubules to the nucleus. Most tubules are seen in longitudinal section. x 51 000. PLATE :< 51 600.
13. Aggregate
of irregularly
arranged
tubules,
most
type of subreticulum.
of which
with intertubular x 82 500. is located
are seen in cross
adjacent section.
-_ .--
PLATE 8
et al.
adjacent
Rat
Man;
Desert
t Determination
Polymyositis; Hyperthyroidism Denervation Atrophy
Normal
Coxsackie myocarditis In tissue culture Polymyositis
Myotonic dystrophy Polymyositis
SR = sarcoplasmic reticulum.
tubules;
(1 pt) (1 pt)
iguana
Man (7 pts) Man (4 pts) Man (1 pt) 19 day old fetal mouse Man (3 pts)
Pt = patient; T = T-tubule; reticulum; ER = endoplasmic
between
Heart (endocardial endothelium) Heart (capillary endothelium) Soleus Gastrocnemius
Skeletal
Skeletal (capillary endothelium) Heart (capillary endothelium) Hindlimb
Gastrocnemius
* Center-to-center distance = No data available.
& [SO]
[26]
[51]
etnl.
etal.
ABBREVIATIONS:
-
[49]
et al. [30]
etal.
Pellegrino Franzini
Ferrans
Pham
Gyorkey
Pappas
[31]
[44]
[58]
Haas & Yunis
Norton
Schotland
-
-
-
-
-
-
reticulum;
EDM
Irregular network Hexagonal
Irregular network Rope-like
Irregular network Irregular network Hexagonal
Hexagonal
Clear
EDM
Clear
EDM
Clear
EDM
Clear
-
RER
T
details;
SR
ER
RER
T
ERt
RER
T
endoplasmic
for further
(in some)
= rough
z See reference dense material;
illustrations; = electron
by us from published
250t
1000
1000-l
300-350
250-750
200-220
450t
230
200-250
260-390
Heart
(2 pts)
Man
Bat Cardiac hypertrophy (AI, ASH)
Normal
ABBREVIATIONS:
Pt = patient; T = T-tubule; reticulum; ER = endoplasmic
tissues
570t
430t
500-l
130
600-700
500-830
660-1200
800t
lOOOf
-
100-230 700-1400
1000
Irregular Irregular
6407
590-960
Hexagonal; irregular
Hexagonal
Irregular
Hexagonal
Hexagonal
Irregular
1140t
Hexagonal Irregular
Tubular arrangement
in muscle
Intertubular distance* (4
described
1050t 1500-2000
Overall diameter of tubules (4
tubules
SR = sarcoplasmic reticulum; EDM reticulum; AI = aortic insufficiency;
$ Patients
shown
Amorphous material Spicules and homogeneous EDM
Clear
Granular
Clear
Clear Dense particles (1400 A diameter)
Flecks of EDM
Clear Granular
Content of tubular lumen
= electron dense material; RER = rough ASH = asymmetric septal hypertrophy.
by us from published illustrations; - = No data available. 4).
Local injection of botulinum toxin Cardiac hypertrophy (AI)
Cold injury Familial periodic paralysis Familial hypokalemic periodic paralysis Polymyositis Hypokalemic periodic paralysis with hyperthyroidism Myasthenia gravis
Condition or disease state
non-branching
t Determined * Center-to-center distance between adjacent tubules; different types of aggregated tubules in the same muscle cells (see Table
Present study
With associatedjilaments Cricothyroid Cho et al. [II]
Cardiac
Gastrocnemius Man (1 Pt)
[18]
Duchen
Present study
Man (1 Pt) Mouse
et al. [5]
Bergman
Gastrocnemius
Man (1 pt) Man (2 pts)$
et al. [45]
Rat Man (1 Pt): Man (1 Pt)
Species
of single-walled,
Deltoid Chou [12,13] Bergman et al. [4] Quadriceps and Dunkle et al. [19] femoris
Odor
Muscle
data on aggregates
associated filaments Tibialis anterior Quadriceps femoris Vastus lateralis
3. Morphologic
Without Price et al. [55] Gruner [29]
Authors (Ref. No.)
TABLE
two endoplasmic
to have
SR
T and SR
SR
-
-
-
SR
SR SR
Tubular connections with
et al. [52]
[29]
4. Morpholigic
Vastus medialis (Type II fibers)
Lewis et al. [39]
Man (I pt)
Man (2 PWS
Man (24pts)
Man (1 pt)S Mouse
Specres
tissues
-
6OOt
950t
6OOt
-
of Intertubular distance* (4
in muscle
Diameter tubules (outer/inner) (4
described
Condition or disease state
tubules
Familial periodic 450-500/ 150 paralysis Implantation 500-650/200-300 of benzpyrene Hypokalemic and 700/430 hyperkalemic periodic paralysis and a variety of other conditions$ Hypokalemic 525/300 periodic paralysis with hyperthyroidism 800/400 Presumed acute muscle infarction
of double-walled
Irregular
Hexagonal Irregular
Hexagonal
Hexagonal
Hexagonal
Tubular arrangement
Dense/clear
Clear/clear
Dense flecks of EDM Clear/dense
Clear/clear
Content of tubules (outer/inner)
-II
SR (lateral sacs)
ERTl
SR
Tubular connections with
ABBREVIATIONS:
Pt = patient; reticulum; -
T = T-tubule; SR = = No data available.
sarcoplasmic
reticulum;
EDM
=
electron
dense
material;
ER
=
endoplasmic
* Center-to-center distance between adjacent tubules; $ Patients shown to have two t Determination by us from published illustrations; different types of aggregated tubules in the same muscle cells (see Table 3) ; 5 See reference for further 7 These tubules were interpreted details; as being virus-like particles derived from endoplasmic reticulum; /I These tubules were interpreted as possibly representing abnormal mitochondria.
Quadriceps femoris
Lumbosacral (Macrophages) Skeletal (Type II fibers)
Quadriceps
Muscle
data on aggregates
Bergman et al. [4] and Dunkle etal. [19]
Engel et al. [.?2]
Popoff
Gruner
Authors (Ref. No.)
TABLE
258
B. J. MARON
AND
V. J. FERRANS
and 3). Aggregates of branching, single-walled tubules (Table 2) were further identified depending on whether their components were continuous with the T system [14, 21, 23, 34, 49, 50, 58, 591 or with the sarcoplasmic reticulum [26, 39, 31, 41, 44, 48, 511. Most aggregates of non-branching, single-walled tubules (Table 3) were reported as being derived from components of sarcoplasmic reticulum [29, 45, 551; in one report [II] these aggregates were described as continuous with both the sarcoplasmic reticuIum and the T system. The two types of aggregates described in the present communication belong to this category of non-branching, single-walled tubules derived from the sarcoplasmic reticulum. This constitutes the first description of such structures in cardiac muscle. Aggregates of double-walled tubules (Table 4) have been described in skeletal muscle [4, 19, 22, 29, 39, 521, but not in cardiac muscle; they bear little resemblance to other types of aggregates and are of uncertain origin. Aggregates of single-walled, branching tubules derived from the T system have been described as arranged either irregularly [23] or in highly regular, lattice-like configurations [la, 21, 34, 49, 50, 58, 591, and have been demonstrated to arise from inpocketings of the plasma membranes [34]. Definition of the relation between aggregates of branching tubules and the transverse tubular system has been achieved by demonstrating the passage of ferritin from the extracellular space and T tubules into the lumina of tubules in the aggregates [23, 341. Aggregates of single-walled, branching tubules derived from endoplasmic reticulum have been described most frequently in a number of conditions which are classified as autoimmune diseases, particularly systemic lupus erythematosus [see 30, 57, 60 for reviews] as we11 as in numerous viral infections [see 57 and 60 for reviews]. These aggregates have been studied most extensively in glomerular endothelium. They have not been described in cardiac muscle cells, but have been reported in capillary endothelium of the heart [26, 311. Similar aggregates have been reported in skeletal muscle cells [30] and capillary endothelium [44] of patients with polymyositis. These aggregated tubules have small diameters (200 to 300 A) and short lengths, and are commonly located within cisterns of rough endoplasmic reticulum, but also occur between the inner and the outer nuclear membranes [26] and even within nuclei [IO, 28, 36, 431. They form complex threedimensional networks of tubules, the ultrastructural appearance of which depends on the plane of section [9]. Their pattern of branching varies considerably, and in a number of reports the branching tubules have been shown to form highly regular crystalline aggregates [2, 7, 8, 17, 40, 48, 561. These branching tubules differ clearly from those found in the 3 patients reported in this communication. Although aggregates of branching tubules have been considered by some to have a structural similarity to nucleocapsids of viruses of the myxo- or paramyxovirus group [30], other investigators have shown that myxoviruses and these tubules differ morphologically [GO] and histochemically [57]. It is most likely that aggregates of this type represent a nonspecific response of the endoplasmic reticulum to cellular injury or
AGGREGATES
OF TUBULES
259
viral infection. The characteristic locations of the branching tubules just described serve to differentiate them from proliferating tubules of endoplasmic reticulum which occur free in the cytoplasm and do not become aggregated into discrete masses. The latter type of tubular proliferation can be induced in liver cells by a variety of drugs [16’, 20, 33, 46, 541. It has been our experience that proliferation of sarcoplasmic reticulum, without aggregation of tubules, is a common finding in hypertrophied cardiac muscle cells. Aggregates of long, straight, single-walled tubules were subdivided into 2 categories according to whether or not intertubular filaments were present within the aggregates (Table 2). Aggregates of single-walled tubules without associated filaments have been described in human skeletal muscle in familial [29] and hypokalemic [4, 19, 451 types of periodic paralysis, myasthenia gravis [IS], polymyositis, 112, 131 and in experimental skeletal muscle injury [18, 551. Aggregates of long, straight and unbranched tubules in all of these described studies and in this communication were found only in muscle cells showing evidence of degeneration or atrophy. The aggregates described by Chou [12, 133 clearly differ from all the others in the category of single-walled, straight tubules in that they were very small in diameter (100 to 230 A), were present within nuclei as well as in the cytoplasm, and were considered to be myxovirus-like. With this exception the aggregates of single-walled, straight tubules without associated filaments (Table 3) are morphologically similar to those described in patient No. 3 in this report. These aggregates differed from those found in our patients No. 1 and No. 2 in that they were not associated with Z band material or intertubular filaments and did not show a highly ordered arrangement. Aggregates of tubules found in cardiac muscle cells of patient No. 3 and in skeletal muscle cells of patients in other reports (Table 3) differ morphologically from crystalline aggregates of microtubules [3J. The latter aggregates have been induced in vitro by periwinkle alkaloids in L-strain fibroblasts and in human leukocytes [3], but have not been described in muscle cells. The microtubules in such aggregates appear cylindrical and are of much smaller diameter (270 A) than the straight-walled tubules in striated muscle. The latter tubules also differ morphologically from annulate lamellae, which are a component of many embryonic, germ or tumor cells and consist of parallel paired membranes (300 to 500 A apart), interrupted at regular intervals by pores [371. Our interpretation of the nature of the tubules in patient No. 3 are that they are greatly elongated, moderately dilated components of sarcoplasmic reticulum. We conclude that these tubules represent an unusual type of proliferation of sarcoplasmic reticulum in cardiac muscle cells that have normally arranged contractile elements. The aggregatesof single-walled tubules associatedwith intertubular filamentous material, which we observed in degenerated cardiac muscle cells from patients No. 1 and No. 2, resemblethosedescribed in cricothyroid muscleof bats [II]. The latter is the only report of aggregated tubules in normal muscle cells. The aggregated tubules in bat skeletal muscle differed significantly from those in our patients in
260
B. J. MARON
AND
V. J. FERRANS
that they had connections with both the sarcoplasmic reticulum and the T system. Furthermore, these tubules and their intertubular filaments were not associated with either normal or abnormal Z band material. Ribosomes are the only other type of structure reported to occur in intertubular spaces of aggregated tubules. Such aggregates, which have been described only in chief cells of bat gastric mucosa [35], were arranged in hexagonal lattices and were composed of long, straight tubules of rough-surfaced endoplasmic reticulum. The tubules found in myocardium of patients No. 1 and No. 2 clearly differ from several types of double-walled tubules that have been reported principally in human skeletal muscle myopathies (Table 4). The exact origin of these double-walled tubules is unclear. According to Engel et al. [.?,?I th ese tubules are connected to the sarcoplasmic reticulum and arise from proliferation of its components. Lewis et al. [39] believe, however, that they are derived from mitochondria. Our concept of the morphogenesis of the hexagonally arranged aggregates of tubules found in two of the patients (Nos. 1 and 2) in this report is illustrated in Plates 9 to 11. Initially, marked focal proliferation and dilatation of the sarcoplasmic reticulum occurs in association with large masses of abnormal Z band material to which thin (actin) filaments are attached. In the early stages of formation of the aggregates, the tubules of sarcoplasmic reticulum appear as irregularly shaped cylinders surrounded by Z band material and actin filaments, and the distance between adjacent tubules is not uniform. These tubules appear to be precursors of the hexagonally arranged tubules in larger aggregates which are not associated with Z band material. As the mass of Z band material surrounding the tubules disappears, the tubules become more cylindrical, more closely associated and evenly spaced. The intertubular filaments and associated electron-dense material probably represent portions of the actin filaments and masses of Z band material previously present in the intertubular spaces. A close relationship was evident between developing aggregates of tubules and abnormal myofibrils composed of irregularly-shaped masses of Z band material and associated thin myofilaments. The masses of abnormal Z band material observed in patients Nos 1 and 2 resemble those described by Munnell and Getty [42] in ventricular myocardium from old dogs, by C&C et al. [15] in atria1 muscle from patients with rheumatic heart disease and by Legato [38] in atria1 and ventricular muscle from patients with a variety of congenital cardiac lesions and in embryonic rat ventricular muscle grown in tissue culture. In all these instances the Z band material showed a periodic arrangement due to the presence of filaments that were spaced 200 A apart and were oriented parallel to the longitudinal axis of the myofibrils. Such an arrangement of Z band material differs from that described by Fawcett [24] in myocardium from old cats and by Bishop and Cole [S] in hypertrophied and failing canine myocardium in that it lacks the well defined transverse (i.e., perpendicular to the longitudinal axes of the myofibrils) periodicity of about 200 A found in the Z band material observed by these authors. The significance of
AGGREGATES
OF TUBULES
261
these differences in arrangement of Z band material is unclear. Legato has postulated [38] that focal accumulations of Z band material represent early stages in the formation of new sarcomeres during normal growth or hypertrophy of cardiac muscle cells. We believe that the masses of Z band material in abnormal myofibrils which lacked thick myofilaments are indicative of degeneration rather than of hypertrophy. This degeneration was characterized by selective loss of thick (myosin) myofilaments from the cardiac muscle cells and was present in variable degrees of severity in biopsies from 13 of the 24 patients in this study. Only in 2 of these 13 patients, however, did we find aggregates of tubular structures associated with intertubular material. These observations are consistent with the hypothesis that such aggregates of tubules form only in those cardiac muscle cells in which the sarcoplasmic reticulum is still capable of proliferating after this type of myofibrillar degeneration develops. It is not known whether Z bands, T tubules and thick myofilaments play a direct role in inducing the normal morphogenesis of sarcoplasmic reticulum components which either surround the myofibrils (longitudinal elements) or are closely associated with Z bands and T tubules (terminal cisterns). The observations in this report suggest that in the presence of advanced degenerative changes (i.e., abnormal masses of Z band material and absence of T tubules and thick myofilaments) growth of the sarcoplasmic reticulum proceeds in a highly abnormal fashion. Data from other studies suggest that this is also the case in skeletal muscle cells, as evidenced by reports of the presence of isolated masses of thin myofilaments in cells containing aggregates of tubules [.5, 4.51. Thus, we conclude that the formation of aggregates of tubules derived from the sarcoplasmic reticulum is a degenerative change related to the inability of the sarcoplasmic reticulum to form meshworks around abnormally constituted myofibrils, or terminal cisterns around T tubules. REFERENCES 1. 2. 3. 4. 5. 6.
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