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Ultrastructural changes in the cardiomyopathy of dystrophic hamsters and mice
TISSUE 0
& CELL
198X Longman
1988 20 (2) 249-253 Group
UK
Ltd
MELINDA
F. RUDGE and C. J. DUNCAN
ULTRASTRUCTURAL CHANGES IN THE CARDIOMYOPATHY OF DYSTROPHIC HAMSTERS AND MICE Keywords:
Cardiomyopathy,
ABSTRACT.
Changes
dystrophic
the
myofilament (including the
swelling,
raised.
of skeletal
implicated
(22-W
mwchondna.
of the cardiac
muscle
muscle
damage
cells have been
damage
skeletal
of
the
and apparent muscle
confirming and cardiac
in the degradative
There is currently considerable interest in Duchenne muscular dystrophy and yet the underlying biochemical pathways and cellular events of the damage to the skeletal muscle cells remain to be elucidated. Parallel studies with incubated preparations have shown that rises in intracellular calcium concentration ([Cal,) can produce characteristic damage in vertebrate skeletal muscle by at least two pathways; one produces the specific degradation of the myofilament apparatus (Duncan. 1987) whilst the other causes sarcolemma breakdown, probably via lipoxygenase activation (Duncan and Jackson. 1987). Animal dystrophies (in mouse, hamster and chicken) have provided suitable models for the study of dystrophic muscle and, again. changes in [Cal, have been implicated in the underlying cellular events (Bhattacharya et ul., 1YX2). In the dystrophic hamster there is an accompanying cardiomyopathy (Jasmin and Solymoss, 1975) and it has been suggested that both myopathies are the consequence of the same gene defect (Homburger e/ ul.. 1970). In the present paper the ultra-
and
skeletal
muscle.
division)
The
degradativc
are characteristic
of normal
the suggestions muscle
changes
Introduction
followed
m
weeks of age) and in both species a severe cardiomyopathy changes
of the heart cells and the specific changes in mitochondrial
septation
dystrophic
cxperimcntally myopathy
cellular
apparatus
calcium.
in the ultrastructure
mice and hamsters
accompanies
both
dystrophy,
that
m animal
of muscle
cardiac
muscle
of the cellular in which
damage
[Cal,
and (ii)
rhe
that change\
of
has hcen
(i) the same gene IS responsible
dystrophy
ut
ultrastructurc
tcrr the
in l(‘a[
.LIC
cc&,
structural changes in cardiac muscle have been followed during maturation in murine and hamster dystrophies and compared with experimentally induced damage in normal mouse skeletal and cardiac muscles and with the skeletal muscles of dystrophic mice and hamsters. Materials and Methods Dystrophic hamsters. in-bred strain CA and CA (albino) controls were obtained from Intersimian Ltd, Milton Trading Estate. Milton, Abington, Oxon. Dystrophic mice. strain CS7BL/6J were provided by courtesy of Dr A. C. Wareham and a breeding stock was established from known heterozypote parents, backcrosses being carried out to establish further heterozygotes. Animals were killed by cervical dislocation and tissue samples were washed in KrehsHenseleit solution and fixed in Karnovsky fixative at room temperature. Subsequent treatment for electron microscopy has heen described previously (Rudge and Duncan. 1984a). Results
Univcrsit> Box
01 L.iverpool.
147. Lwxpool
RCWlVCd
Department
Lh9 3BX.
23 Octohcr
IYX7.
of Zoology.
PO
Tissue samples were examined from the hearts of dystrophic mice and Syrian hams-
RUDGE AND DUNCAN
250
ters at 22,28, 34 and 40 weeks of age. Ultrastructural damage of the cardiac muscle cells was found in both species, increasing progressively in extent with age. The ultrastructural changes to the myofilament apparatus (see Figs 1-4, hamster; Figs 5-8, mouse) in dystrophic animals could be classified as: 1. Z-line sliding (Fig. 3) 2. Breaking-up of the Z-line (Fig. 4) culminating in the complete loss of the Z-line (Fig. 4) 3. Separation of the fibrils (Fig. 1) 4. Loss of myofilaments, beginning in focal areas (Figs 5,6), but increasing to show widespread degradation (Figs 2, 4). In some areas damage is concentrated on the actin filaments (Fig. 3), particularly associated with Z-line damage, and in other sections of the muscle the myosin filaments are the main targets (Fig. 2) 5. Contraction of the fibril with widened, blurred Z-lines (Figs 2, 7, 8) 6. Areas of hypercontraction. Accompanying this degradation of the myofilaments, the mitochondria were frequently swollen, with reduced matrix density, together with an increase in mitochondrial numbers, some of which showed the development of internal septa which apparently subdivided the mitochondria (Figs 7, 8). Skeletal muscles from the leg and diaphragm of dystrophic animals were also routinely examined at the different ages, and ultrastructural changes and damage were also
found. The patterns of this damage appeared to be very similar to (if not identical with) those recorded in the cardiac muscle of dystrophic animals (above) and also to the changes previously described in isolated preparations of mouse diaphragm (Duncan et al., 1980) or mouse soleus muscle (Duncan and Jackson, 1987 ) when these have been subjected to various treatments that are believed to raise [Cali in the muscle cells. Discussion This ultrastructural study demonstrates that in the dystrophic mouse, as well as in the dystrophic hamster, there is a severe cardiomyopathy that accompanies the typical myopathy of the skeletal muscle. An analysis of the different types of degradative effects, i.e. damage associated with contraction, as well as the dissolution of the myofilament apparatus and Z-lines in relaxed sarcomeres, together with the highly characteristic changes in the mitochondria (Duncan et al., 1980; Duncan, 1988) which correspond closely with that found in the skeletal muscle of the same animals suggests that the same degradative mechanisms are involved in the myopathies of both cardiac and skeletal muscle, and supports the proposal (Homburger et al. 1970) that the same genetic defect is responsible for the lesions in both tissues. The details of the degradative changes in the hearts of dystrophic mice and hamsters are identical with those described previously in isolated preparations from the hearts of normal mice that had been exposed to various treatments, including the calcium
Figs l-4. Electron micrographs of ventricular tissue from hearts of 34-week-old dystrophic Syrian hamsters. Figs 1, 2: myofibrils are contracted, with myofilament disarray and loss (arrows) and blurred Z-lines. Myofibtils are clearly separated in places (M). The sarcoplasmic ground substance has reduced electron density. Fig. 1, x5ooO; Fig. 2, ~22,000. Figs 3 and 4 show myofilament and Z-line (Z) loss and also Z-line sliding(S). Fig. 3 shows loss of actin filaments in the I-band (I). The sarcoplasmic ground substance has a reduced electron density in both figures and the interfibrillar space is enlarged. Fig. 3, xZO,OC@;Fig. 4, ~20,000. Figs 58. Electron micrographs of ventricular tissue from hearts of 34-week-old dystrophic Figs 5,6: myofibrils are relaxed but focal areas of myofilament loss are apparent (arrows). Fig. 5, X12,m; Fig. 6, X12,500. Figs 7 and 8 show contracted myofibrils with blurred Z-lines. The mitochondria are numerous with a reduced matrix density. Fig. 7, x 12,000; Fig. 8, ~29,000. mice.
CARDIOMYOPATHY
IN ANIMAL
DYSTROPHY
253
paradox, that are believed to raise [Cal,. In particular, the numbers of mitochondria have been determined quantitatively on a microcomputer from planimetric measurements of electron micrographs (Rudge and Duncan, 1984a), and in isolated strips from normal mouse cardiac muscle, exposure to the divalent cation ionophore A23187, for example, not only increased the absolute numbers of mitochondria per cell but also changed the ratio of myofibrillar:mitochondrial areas from 2:l to a condition where the total area occupied by mitochondria exceeded the myofibrillar area. Since these characteristic mitochondrial changes, swelling, septation and subdivision, are also found in dystrophic mice and hamsters, it supports (Rudge and Duncan, 1984a, b) the suggestions that elevated [Cali is implicated in the pathogenesis of both the cardiomyopathy of dystrophic mice and hamsters and also of dystrophic mammalian skeletal muscle (Duncan, 1978).
Thus, dystrophic hamsters have been treated with Ca-channel blockers; verapamil prevented necrosis of cardiac muscle while cinnarizine reduced the severity of myocardial lesions by more than 50%; diltiazem produced a reduction in cardiac muscle Ca and plasma creatine kinase fell (Bhattacharya et af., 1982)) although little beneficial result was found in skeletal muscle with any treatment (Cosmos and Butler, 1980). A Ca-deficient diet reduced the severity of the lesions in both cardiac and skeletal muscle (Jasmin and Solymoss, 1975). In malignant hyperthermia, a genetic disease of man and pigs, halothane anaesthesia causes the impairment of the functioning of the sarcoplasmic reticulum of skeletal muscle, a clearly established elevation of [Cal, (Lopez et al., 1985, 1987) and severe muscle damage. It is noteworthy that cardiac muscle is also involved in humans suffering from malignant hyperthermia (Britt . 1974).
References Bhattacharya, S. K., Palmieri, G. M. A., Bertorini, T. E. and Nutting, D. F. 1982. Effect of the calcium antagomst diltiazem in dystrophic hamsters. Muscle and Nerve, 5, 73-78. Britt, B. A. 1974. Malignant hyperthermia. A pharmacogenetic disease of skeletal and cardiac muscle. New Eq[_ J Med., 290, 114&1142. Cosmos, E. and Butler. J. 1980. Animal models of muscle diseases, Part III: Compilation of therapeutic trials IOI hereditary muscular dystrophy. Muscle and Nerve, 4, 427-435. Duncan. C. J. 1978. Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophlc condition. Experienrio, 34, 1531-1535. Duncan, C. J. 1987. Role of calcium in triggering rapid ultrastructural damage m muscle: a study with chemically skmned fibrcs. J. Cel[ Sci., 87, 581-594. Duncan. C. J. 1988. Mitochondrial division in animal cells. In S. E. B. Seminar Series. ~0135. The Division of Segregation of Organelles (in press). Duncan. C. J., Greenaway. H. C., Publicover, S. J., Rudge, M. F. and Smith, J. L. 1980. Experimental productjon of ‘septa’ and apparent subdivision of muscle mitochondria. J. Bioenerg. Biomembr., 12, I?-33. Duncan. C. J., Greenaway. H. C. and Smith, J. L. 1980. 2,CDinitrophenol, lysosomal breakdown and rapid myofdament degradation in vertebrate skeletal muscle. Naunyn-SchmiedeberRs. Arch. exp. Parh. Pharmac.. 315, 77-X? Duncan, C. J. and Jackson, M. J. 1987. Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle. J. Cell. Sci.. 87, 18%188. Hornburger, F., Bajusz, E. and Nixon, C. W. 1970. New modelsof human disease in Syrian hamsters .I. Am Med. AA.? 212, 60~610. Jasmin. G. and Solymoss, B. 1975. Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents. Proc. Sot. Exp. Biol. Med., 149, 193-198. Lopez, R. L., Alamo, L., Caputo, C., Wikinski, J. and Ledezma. D. 1985. Intracellularionizedcalcium concentration in muscles from humans with malignant hyperthermia. Muscle and Nerve. 8,355-35X. L.oper, J. R.. Allen, P., Alamo. L., Jones, D. and Sreter, F. 1988. Myoplasmlc free [Ca*-1, durmg a mahgnant hyperthermia episode in swine. Muscle and Nerve, 11, 82-88. Rudge. M. F. and Duncan, C. J. 1984a. Comparative studies on the role of calcium m triggermg subcellular damage in cardiac muscle. Camp. Biochem. Physiok, 77A, 459-468. Rudge, M. F. and Duncan, C. J. 1984b. Comparative studies on the calcium paradox m cardiac muscle: the ettect of temperature on the different phases. Camp. Biochem. Physiol., 79A, 393-398.
& CELL
198X Longman
1988 20 (2) 249-253 Group
UK
Ltd
MELINDA
F. RUDGE and C. J. DUNCAN
ULTRASTRUCTURAL CHANGES IN THE CARDIOMYOPATHY OF DYSTROPHIC HAMSTERS AND MICE Keywords:
Cardiomyopathy,
ABSTRACT.
Changes
dystrophic
the
myofilament (including the
swelling,
raised.
of skeletal
implicated
(22-W
mwchondna.
of the cardiac
muscle
muscle
damage
cells have been
damage
skeletal
of
the
and apparent muscle
confirming and cardiac
in the degradative
There is currently considerable interest in Duchenne muscular dystrophy and yet the underlying biochemical pathways and cellular events of the damage to the skeletal muscle cells remain to be elucidated. Parallel studies with incubated preparations have shown that rises in intracellular calcium concentration ([Cal,) can produce characteristic damage in vertebrate skeletal muscle by at least two pathways; one produces the specific degradation of the myofilament apparatus (Duncan. 1987) whilst the other causes sarcolemma breakdown, probably via lipoxygenase activation (Duncan and Jackson. 1987). Animal dystrophies (in mouse, hamster and chicken) have provided suitable models for the study of dystrophic muscle and, again. changes in [Cal, have been implicated in the underlying cellular events (Bhattacharya et ul., 1YX2). In the dystrophic hamster there is an accompanying cardiomyopathy (Jasmin and Solymoss, 1975) and it has been suggested that both myopathies are the consequence of the same gene defect (Homburger e/ ul.. 1970). In the present paper the ultra-
and
skeletal
muscle.
division)
The
degradativc
are characteristic
of normal
the suggestions muscle
changes
Introduction
followed
m
weeks of age) and in both species a severe cardiomyopathy changes
of the heart cells and the specific changes in mitochondrial
septation
dystrophic
cxperimcntally myopathy
cellular
apparatus
calcium.
in the ultrastructure
mice and hamsters
accompanies
both
dystrophy,
that
m animal
of muscle
cardiac
muscle
of the cellular in which
damage
[Cal,
and (ii)
rhe
that change\
of
has hcen
(i) the same gene IS responsible
dystrophy
ut
ultrastructurc
tcrr the
in l(‘a[
.LIC
cc&,
structural changes in cardiac muscle have been followed during maturation in murine and hamster dystrophies and compared with experimentally induced damage in normal mouse skeletal and cardiac muscles and with the skeletal muscles of dystrophic mice and hamsters. Materials and Methods Dystrophic hamsters. in-bred strain CA and CA (albino) controls were obtained from Intersimian Ltd, Milton Trading Estate. Milton, Abington, Oxon. Dystrophic mice. strain CS7BL/6J were provided by courtesy of Dr A. C. Wareham and a breeding stock was established from known heterozypote parents, backcrosses being carried out to establish further heterozygotes. Animals were killed by cervical dislocation and tissue samples were washed in KrehsHenseleit solution and fixed in Karnovsky fixative at room temperature. Subsequent treatment for electron microscopy has heen described previously (Rudge and Duncan. 1984a). Results
Univcrsit> Box
01 L.iverpool.
147. Lwxpool
RCWlVCd
Department
Lh9 3BX.
23 Octohcr
IYX7.
of Zoology.
PO
Tissue samples were examined from the hearts of dystrophic mice and Syrian hams-
RUDGE AND DUNCAN
250
ters at 22,28, 34 and 40 weeks of age. Ultrastructural damage of the cardiac muscle cells was found in both species, increasing progressively in extent with age. The ultrastructural changes to the myofilament apparatus (see Figs 1-4, hamster; Figs 5-8, mouse) in dystrophic animals could be classified as: 1. Z-line sliding (Fig. 3) 2. Breaking-up of the Z-line (Fig. 4) culminating in the complete loss of the Z-line (Fig. 4) 3. Separation of the fibrils (Fig. 1) 4. Loss of myofilaments, beginning in focal areas (Figs 5,6), but increasing to show widespread degradation (Figs 2, 4). In some areas damage is concentrated on the actin filaments (Fig. 3), particularly associated with Z-line damage, and in other sections of the muscle the myosin filaments are the main targets (Fig. 2) 5. Contraction of the fibril with widened, blurred Z-lines (Figs 2, 7, 8) 6. Areas of hypercontraction. Accompanying this degradation of the myofilaments, the mitochondria were frequently swollen, with reduced matrix density, together with an increase in mitochondrial numbers, some of which showed the development of internal septa which apparently subdivided the mitochondria (Figs 7, 8). Skeletal muscles from the leg and diaphragm of dystrophic animals were also routinely examined at the different ages, and ultrastructural changes and damage were also
found. The patterns of this damage appeared to be very similar to (if not identical with) those recorded in the cardiac muscle of dystrophic animals (above) and also to the changes previously described in isolated preparations of mouse diaphragm (Duncan et al., 1980) or mouse soleus muscle (Duncan and Jackson, 1987 ) when these have been subjected to various treatments that are believed to raise [Cali in the muscle cells. Discussion This ultrastructural study demonstrates that in the dystrophic mouse, as well as in the dystrophic hamster, there is a severe cardiomyopathy that accompanies the typical myopathy of the skeletal muscle. An analysis of the different types of degradative effects, i.e. damage associated with contraction, as well as the dissolution of the myofilament apparatus and Z-lines in relaxed sarcomeres, together with the highly characteristic changes in the mitochondria (Duncan et al., 1980; Duncan, 1988) which correspond closely with that found in the skeletal muscle of the same animals suggests that the same degradative mechanisms are involved in the myopathies of both cardiac and skeletal muscle, and supports the proposal (Homburger et al. 1970) that the same genetic defect is responsible for the lesions in both tissues. The details of the degradative changes in the hearts of dystrophic mice and hamsters are identical with those described previously in isolated preparations from the hearts of normal mice that had been exposed to various treatments, including the calcium
Figs l-4. Electron micrographs of ventricular tissue from hearts of 34-week-old dystrophic Syrian hamsters. Figs 1, 2: myofibrils are contracted, with myofilament disarray and loss (arrows) and blurred Z-lines. Myofibtils are clearly separated in places (M). The sarcoplasmic ground substance has reduced electron density. Fig. 1, x5ooO; Fig. 2, ~22,000. Figs 3 and 4 show myofilament and Z-line (Z) loss and also Z-line sliding(S). Fig. 3 shows loss of actin filaments in the I-band (I). The sarcoplasmic ground substance has a reduced electron density in both figures and the interfibrillar space is enlarged. Fig. 3, xZO,OC@;Fig. 4, ~20,000. Figs 58. Electron micrographs of ventricular tissue from hearts of 34-week-old dystrophic Figs 5,6: myofibrils are relaxed but focal areas of myofilament loss are apparent (arrows). Fig. 5, X12,m; Fig. 6, X12,500. Figs 7 and 8 show contracted myofibrils with blurred Z-lines. The mitochondria are numerous with a reduced matrix density. Fig. 7, x 12,000; Fig. 8, ~29,000. mice.
CARDIOMYOPATHY
IN ANIMAL
DYSTROPHY
253
paradox, that are believed to raise [Cal,. In particular, the numbers of mitochondria have been determined quantitatively on a microcomputer from planimetric measurements of electron micrographs (Rudge and Duncan, 1984a), and in isolated strips from normal mouse cardiac muscle, exposure to the divalent cation ionophore A23187, for example, not only increased the absolute numbers of mitochondria per cell but also changed the ratio of myofibrillar:mitochondrial areas from 2:l to a condition where the total area occupied by mitochondria exceeded the myofibrillar area. Since these characteristic mitochondrial changes, swelling, septation and subdivision, are also found in dystrophic mice and hamsters, it supports (Rudge and Duncan, 1984a, b) the suggestions that elevated [Cali is implicated in the pathogenesis of both the cardiomyopathy of dystrophic mice and hamsters and also of dystrophic mammalian skeletal muscle (Duncan, 1978).
Thus, dystrophic hamsters have been treated with Ca-channel blockers; verapamil prevented necrosis of cardiac muscle while cinnarizine reduced the severity of myocardial lesions by more than 50%; diltiazem produced a reduction in cardiac muscle Ca and plasma creatine kinase fell (Bhattacharya et af., 1982)) although little beneficial result was found in skeletal muscle with any treatment (Cosmos and Butler, 1980). A Ca-deficient diet reduced the severity of the lesions in both cardiac and skeletal muscle (Jasmin and Solymoss, 1975). In malignant hyperthermia, a genetic disease of man and pigs, halothane anaesthesia causes the impairment of the functioning of the sarcoplasmic reticulum of skeletal muscle, a clearly established elevation of [Cal, (Lopez et al., 1985, 1987) and severe muscle damage. It is noteworthy that cardiac muscle is also involved in humans suffering from malignant hyperthermia (Britt . 1974).
References Bhattacharya, S. K., Palmieri, G. M. A., Bertorini, T. E. and Nutting, D. F. 1982. Effect of the calcium antagomst diltiazem in dystrophic hamsters. Muscle and Nerve, 5, 73-78. Britt, B. A. 1974. Malignant hyperthermia. A pharmacogenetic disease of skeletal and cardiac muscle. New Eq[_ J Med., 290, 114&1142. Cosmos, E. and Butler. J. 1980. Animal models of muscle diseases, Part III: Compilation of therapeutic trials IOI hereditary muscular dystrophy. Muscle and Nerve, 4, 427-435. Duncan. C. J. 1978. Role of intracellular calcium in promoting muscle damage: a strategy for controlling the dystrophlc condition. Experienrio, 34, 1531-1535. Duncan, C. J. 1987. Role of calcium in triggering rapid ultrastructural damage m muscle: a study with chemically skmned fibrcs. J. Cel[ Sci., 87, 581-594. Duncan. C. J. 1988. Mitochondrial division in animal cells. In S. E. B. Seminar Series. ~0135. The Division of Segregation of Organelles (in press). Duncan. C. J., Greenaway. H. C., Publicover, S. J., Rudge, M. F. and Smith, J. L. 1980. Experimental productjon of ‘septa’ and apparent subdivision of muscle mitochondria. J. Bioenerg. Biomembr., 12, I?-33. Duncan. C. J., Greenaway. H. C. and Smith, J. L. 1980. 2,CDinitrophenol, lysosomal breakdown and rapid myofdament degradation in vertebrate skeletal muscle. Naunyn-SchmiedeberRs. Arch. exp. Parh. Pharmac.. 315, 77-X? Duncan, C. J. and Jackson, M. J. 1987. Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle. J. Cell. Sci.. 87, 18%188. Hornburger, F., Bajusz, E. and Nixon, C. W. 1970. New modelsof human disease in Syrian hamsters .I. Am Med. AA.? 212, 60~610. Jasmin. G. and Solymoss, B. 1975. Prevention of hereditary cardiomyopathy in the hamster by verapamil and other agents. Proc. Sot. Exp. Biol. Med., 149, 193-198. Lopez, R. L., Alamo, L., Caputo, C., Wikinski, J. and Ledezma. D. 1985. Intracellularionizedcalcium concentration in muscles from humans with malignant hyperthermia. Muscle and Nerve. 8,355-35X. L.oper, J. R.. Allen, P., Alamo. L., Jones, D. and Sreter, F. 1988. Myoplasmlc free [Ca*-1, durmg a mahgnant hyperthermia episode in swine. Muscle and Nerve, 11, 82-88. Rudge. M. F. and Duncan, C. J. 1984a. Comparative studies on the role of calcium m triggermg subcellular damage in cardiac muscle. Camp. Biochem. Physiok, 77A, 459-468. Rudge, M. F. and Duncan, C. J. 1984b. Comparative studies on the calcium paradox m cardiac muscle: the ettect of temperature on the different phases. Camp. Biochem. Physiol., 79A, 393-398.