Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice

A Ca²⁺-activated neutral protease activity was examined in muscles of normal and dystrophic hamsters and mice. Light grey and golden brown strains of normal and B10 14.6 strain of dystrophic hamsters were used. Normal and dystrophic mice were of the Bar Harbor 129 ReJ strain. Enzyme activity was measured in the post myofibrillar fraction (homogenate) and in the 75,000 × g pellet (particulate fraction) and supernatant using purified myofibrils.

In normal and dystrophic hamsters or mice, the Ca²⁺-activated neutral protease was most active in the supernatant followed by the homogenate and particulate fractions. As compared to fractions from normal muscle, enzyme activity was significantly elevated in all 3 fractions from dystrophic muscles of hamsters and mice. Both homogenate and supernatant fractions from muscles of normal hamsters had significantly higher enzyme activity than those of normal mice. Enzyme activity was similar in the particulate fraction. Similarly enzyme activity in the 3 fractions from dystrophic hamster and mouse muscles showed no significant difference.

It is suggested that the Ca²⁺-activated neutral protease may be involved in muscle fibre necrosis in muscular dystrophy.     

An endogenous inhibitor of calcium-activated neutral protease in UMX 7.1 hamster dystrophy

An endogenous inhibitor for calcium-activated neutral protease (CANP) from skeletal and cardiac muscles of muscular dystrophic hamsters (UMX 7.1) was compared with that from normal control animals at 4 and 10 weeks of age by Western blotting using antibody raised against CANP inhibitor. Fragmented CANP inhibitor was found in dystrophic skeletal muscles in all cases at both ages, while only intact inhibitor was detected in the skeletal muscle of the normal hamsters. A total absence of intact inhibitor was shown in one 10-week-old dystrophic hamster. In contrast, there was little difference in CANP inhibitor from heart between dystrophic and control hamsters at 4 weeks. However, fragmentation similar to that in skeletal muscle was seen in the heart inhibitor in a few of the 10-week-old dystrophic hamsters.     

Altered tissue carnitine levels in animals with hereditary muscular dystrophy

Low levels of muscle carnitine have been found in patients with Duchenne dystrophy, a case possibly of Becker dystrophy, and limb-girdle syndrome as well as in patients with the recently described muscle carnitine deficiency syndrome. Tissues of the mouse, hamster, and chicken were analyzed to determine whether tissue carnitine levels were altered in the animal models of muscular dystrophy. Significantly higher levels of carnitine were found in dystrophic mouse muscle, but carnitine levels in plasma, liver and heart were normal. Histological changes in the skeletal muscle of dystrophic hamsters were relatively mild, and both skeletal muscle and plasma levels were normal. The liver carnitine level was higher than normal levels. The dystrophic hamster also had an inherited cardiomyopathy, and interestingly its heart carnitine level was much lower than normal. The red muscle of the normal chicken contained 5 times the level of carnitine found in white muscle. The dystrophic chicken had higher than normal levels of carnitine in the white muscle, but normal levels in the red muscle. Although all 3 animal models of muscular dystrophy studied have altered levels of carnitine in some tissue, none of the animal models had the same pattern of altered tissue carnitine levels seen in human patients.     

Age-related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ.

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.     

Calmodulin content and Ca-activated protease activity in dystrophic hamster muscles

As part of a study on the implication of elevated Ca²⁺ levels in the myofibrillar degeneration seen in dystrophic muscle, the content of calmodulin and the activity of Ca²⁺ -activated neutral protease (CANP) have been measured in normal and dystrophic (UM-X7.1) hamsters. Calmodulin levels, expressed as micrograms ± SEM per gram wet weight were highest in brain (385 ± 24.7), followed by tongue (93.88 ± 3.93), heart (42.13 ± 2.93), and skeletal muscle (31.69 ± 1.42). No significant increases in calmodulin were observed in the dystrophic tissues thus suggesting that the Ca²⁺ accumulations observed in dystrophic muscles are unrelated to changes in calmodulin levels. Because of the complexity of regulation of CANP, a time-dependent study was done using extracts of skeletal, heart, and tongue muscles. Marginal increases in dystrophic CANP were seen in skeletal muscle at all times studied and in the heart and tongue at initial time points only. The data are discussed in terms of rising levels of Ca²⁺ in muscles of the UM-X7.1 hamster being sufficient to increase CANP activity (without increasing content) to where it causes Z-line dissolution.     

Serum CK,calcium, magnesium,and oxidative phosphorylation in mdx mouse muscular dystrophy

Serum creatine kinase (CK) activity, calcium (Ca) and magnesium (Mg) contents of skeletal muscle and isolated mitochondria, as well as oxidative phosphorylation of X-linked muscular dystrophic (mdx) mice were compared with normal control animals at ages 5, 10, and 23 weeks. Serum CK is elevated in mdx mice at all ages, with highest activities at 5 weeks. The Ca content of dystrophic skeletal muscle is increased at all ages, whereas no clearly abnormal trend in muscle Mg levels was observed. Noncollagen protein (NCP), which was used as a reference base, is significantly diminished in muscle from 10- and 23-week-old mdx animals. Isolated mitochondria from mdx mice have elevated calcium content and decreased respiratory control ratios with NAD-linked substrates pyruvate/malate. The findings are distinct from those in dystrophic mice, strain 129/ReJ, but similar to observations in dystrophic hamsters and Duchenne muscular dystrophy and reflect the occurrence of overt muscle cell necrosis.     

Glyceraldehyde-3-phosphate dehydrogenase mRNA. Activity and amount in dystrophic hamster muscle

The activity and amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in muscle of young dystrophic hamsters was reduced to approximately half the level found in control animals. No changes in brain or liver enzyme activity were found. Several other glycolytic enzyme activities and creatine kinase activity in muscle were unchanged, except for modest decreases in aldolase and pyruvate kinase. To assess the synthesis of glyceraldehyde-3-phosphate dehydrogenase, the poly(A)+ RNA was isolated from muscle polysomes of dystrophic and control animals and its activity was assessed in an mRNA-dependent translation system. The translatability of the mRNA for GAPDH found in the dystrophic muscle preparations also was half of that found in the control muscle preparations. Decreases were also found in the translatability of mRNA for tropomyosin.     

Lipid peroxide and antioxidant enzymes in muscle and nonmuscle of dystrophic mouse

To determine whether abnormality in redox metabolism occurs specifically in certain individual dystrophic muscles, thiobarbituric acid reactivity, free radical scavengers, and oxidative marker enzymes were measured in the liver, kidney, erythrocytes, heart, and four different individual skeletal muscles from C57BL/6J dy/dy mice. Superoxide dismutases were assayed by specific radioimmunoassays, which enabled the study of a small individual murine muscle. Glutathione peroxidase and catalase were increased markedly in each individual dystrophic skeletal muscle studied and less markedly in the heart. Manganosuperoxide dismutase and thiobarbituric acid reactivity were decreased to a similar extent in each dystrophic skeletal muscle. Cuprozinc superoxide dismutase was decreased in the soleus muscle. Only a minimal biochemical change occurred in nonmuscles. Fumarase activity correlated closely with the level of manganosuperoxide dismutase. These results suggest that muscle protein breakdown occurs independently of lipid peroxidation despite the presence of tissue-specific abnormality of redox metabolism in dystrophic muscle.     

Myofibrillar M-line structure in normal and dystrophic hamster muscle

The presence of the specific myofibrillar M-line marker, myomesin , in isolated myofibrils and cryosections of skeletal and heart muscle as well as its appearance during differentiation in skeletal and heart muscle cell cultures of normal and dystrophic hamsters were evaluated. By means of the indirect immunofluorescence technique employing antibodies against chicken M-line proteins, the appearance of antigen localized in the M-line was investigated. No difference could be found between the M-line structure of normal and dystrophic animals. The results suggest that the M-line proteins, apparently relatively stable, are not primarily affected by the disease.     

Ouabain binding sites in skeletal muscle from normal and dystrophic mice.

The specific binding of tritiated ouabain was used to estimate the density of Na+-K+-ATPase sites ("Na+-pump" sites) in segments of skeletal muscle from normal and dystrophic mice. Ouabain binding was approximately 4 times greater in red (soleus) muscle than in white (superficial gastrocnemius) muscle from normal animals. In dystrophic soleus muscles, ouabain binding was decreased by nearly one-half. Because Na+-K+-ATPase activity is associated with plasma membranes, these observations constitute further evidence for a sarcolemmal abnormality in dystrophic mice.     

Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle of dystrophic mice

Mitochondrial calcium overloading was investigated in the genetically dystrophic mouse (strains $\text{129}\text{ReJ}\text{dy}\text{dy}$) as a possible contributing factor to the development of muscle fiber necrosis. Mitochondrial calcium concentrations were significantly elevated in both skeletal muscle and heart organelles. Because mitochondria were isolated in the presence of ruthenium red this finding was not the result of an artefact of isolation. State 3 respiration rates and concomitantly the respiratory control ratios were slightly decreased in skeletal muscle, but not in heart mitochondria. This abnormality could result from calcium overloading in a small fraction of the mitochondria. Fractionation of skeletal muscle mitochondria on sucrose gradients gave two distinct populations of dystrophic organelles, one with high calcium, whereas normal skeletal muscle mitochondria and heart organelles showed only one broad band on the gradient. The results support the idea that both skeletal muscle and heart are affected in dystrophic mice, strain $\text{129}\text{ReJ}\text{dy}\text{dy}$ and also that in the dystrophic mouse the process of cell necrosis is associated with cellular calcium overloading.     

The effect of age on the nucleic acid content of slow- and fast-twitch muscle in normal and dystrophic mice and their litter mates

RNA, DNA, and NCP content were measured in fast- and slow-twitch skeletal muscle of normal and dystrophic mice (HDM) and their littermates at ages 4 through 29 weeks.In normal and litter mate mice RNA and DNA content were far greater in the soleus than in the gastrocnemius while the RNA/DNA ratio and NCP content were greater in the gastrocnemius. In dystrophic mice, however, the differences between nucleic acid content of the 2 muscles were far less, apparently due to a proportionately higher content in the dystrophic gastrocnemius. Due to a proportionately lower ratio in the gastrocnemius, dystrophic RNA/DNA ratios for the 2 muscles were essentially the same.Age had a marked effect on the nucleic acid content of both muscles in all 3 mice types but to varying degrees. In the soleus, RNA and DNA content rapidly decreased until 9 to 10 weeks of age followed by a gradual decline. Soleus RNA/DNA ratios showed little change with age except in the HDM mice in which there was a significant overall decline. In the gastrocnemius, RNA content followed the same pattern but with a smaller decline in the younger ages. Age had no affect on DNA content in the normal gastrocnemius, but there was a significant decline in the HDM gastrocnemius. RNA/DNA gastrocnemius ratios showed marked fluctuations in both normal and dystrophic mice but did not appear to be affected by age.Depending upon age and muscle type, dystrophic RNA content was significantly higher in both muscle types from 5 to 9 weeks of age, and then decreased below normal levels. DNA content of both dystrophic soleus and gastrocnemius was also elevated in the young mice. In the soleus, DNA also decreased below normal values during middle age but then increased again in the middle and older age mice. The RNA/DNA ratio of the dystrophic gastrocnemius was decreased in most age groups. The ratio for the soleus, however, had 3 phases; a reduced ratio in the young mice, a higher ratio in the middle-aged mice, and a reduced ratio in the oldest mice.     

The effects of diltiazem in dystrophic hamsters

Calcium accumulates in muscles of dystrophic hamsters (DH) and patients with Duchenne muscular dystrophy. Various Ca antagonists were beneficial to the cardiomyopathy of DH, but had only minor effects on skeletal muscle. We administered a new Ca antagonist, diltiazem, 25 mg/kg/day orally to normal and dystrophic hamsters from ages 37 to 92 days. We observed a marked reduction in muscle Ca in DH treated with diltiazem: 73% in the heart, 61% in the diaphragm, and 48% in the rectus femoris. Plasma CK was significantly lower (by 37%) in treated DH, while the elevated rate of noncollagen protein synthesis in the diaphragm was not diminished. Histologically, the most important change was a reduction in Ca deposits in the heart. Diltiazem was well-tolerated by all animals and did not modify Ca content in normal hamsters. This study suggests that diltizem may have therapeutic value in those conditions that are accompanied by excessive accumulation of Ca in tissues, such as muscular dystrophy.     

Beneficial effect of chymostatin on dystrophic mice

We studied the effect of chymostatin on dystrophic mice (C57BL/6J-dy). The locomotor activity of normal mice increased markedly, attaining a plateau at 8 weeks of age, whereas in dystrophic mice, it increased until the age of 7 weeks, and thereafter decreased gradually. Serum levels of creatine phosphokinase were significantly higher in dystrophic mice compared with normal mice, and dystrophic mice had a reduced muscle protein content. When 3-week-old dystrophic mice received chymostatin (1 mg/kg, i.p.), the decrease in locomotor activity was retarded, serum enzyme levels decreased significantly, and muscle protein content increased significantly. In addition, the survival time of treated dystrophic mice was prolonged. The locomotor activity, serum enzyme levels, and muscle protein content of normal mice were not affected by chymostatin. Therefore, we posit that chymostatin retarded the progression of dystrophy in mice.     

Choline acetyltransferase and acetylcholinesterase in muscular dystrophic mice

The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in various tissues of dystrophic (dy/dy) and normal mice of Bar Harbor strain 129. The brain weights of these dystrophic mice were not significantly different from those of normal mice, but the average body weight of these dystrophic mice was only 66.8% of that of the controls. The activity of CAT (expressed as unit activity per mg of protein) was very similar in the brains of both groups of animals, but the CAT activity (per mg of protein) in the hindlimb muscles of the dystrophic mice was significantly higher than that of the controls. The patterns of AChE activity, as separated by sucrose density gradient centrifugation, were distinctly different in extracts of dystrophic and normal muscle. Compared with controls, decreased activity of the 15-S and 10-S forms of AChE, with increased activity of a 4.3-S forrn of AChE, was observed in dystrophic muscle.     

On the specificity of the acetylcholinesterase defect in dystrophic mice

The tetrameric form of acetylcholinesterase (AChE) in ReJ/129 dystrophic mice was demonstrated to be absent from endplate-poor regions of skeletal muscle but present in endplate-rich regions. Skeletal muscle secreted normal amounts of this form of AChE. Visceral organs had normal amounts and distribution of the AChE molecular forms. These results suggest that the AChE defect in dystrophic mice is limited to skeletal muscle, and the defect does not reflect an abnormality of AChE synthesis but probably reflects an inability to incorporate the enzyme into skeletal muscle membranes.     

Reduced axonal transport of 10S acetylcholinesterase in dystrophic mice

Extracts of extensor digitorum longus muscle, atria, brain, and sciatic nerve from phenotypically normal and dystrophic ReJ/129 mice were subjected to sucrose density gradient ultracentrifugation, and the amounts of acetylcholinesterase (AChE) activity associated with each major enzyme form were determined. Normal muscle showed approximately equivalent amounts of the 4S, 10S, and 16S forms of AChE, while dystrophic muscle was relatively deficient in 10S AChE and relatively oversupplied with 4S AChE. This abnormality was not present in the other tissues examined. However, as measured by the 24-hour accumulation of enzye activity proximal to a ligature on the sciatic nerve, the axonal transport of 10S AChE was only about one third as great in dystrophic as in normal nerve. This result is consistent with the view that the reduction in the amount of this enzyme form in dystrophic muscle could be related to disturbances in a transport-dependent trophic interaction between nerve and muscle.     

Creatine kinase isozyme transition in chicks with hereditary muscular dystrophy

In both normal chicks and chicks with hereditary muscular dystrophy the BB (brain) and MB (hybrid) isozymes were the predominant forms of creatine kinase (CK) activity in embryonic skeletal muscle. As myogenesis progressed, activity due to the MM (muscle) isozyme progressively increased, and by 1 week ex ovo, the MM isozyme accounted for approximately 97% of total muscle activity in both genotypes. During this time, the proportion of the MM isozyme was slightly but significantly lower in dystrophic muscles. After hatching the proportion of the MB isozyme and its total activity decreased in normal muscle, but increased in dystrophic pectoral muscle, and by 5 months ex ovo, the MB isozyme accounted for 10% of total CK activity. Prior to hatching there was no consistent difference in total CK activity between normal and dystrophic tissues, but by 1 week after hatching and thereafter, total CK activity was significantly lower in dystrophic pectoral muscle.     

Adult-onset acid maltase deficiency: A postmortem study

In a postmortem study of a patient with adult-onset acid maltase deficiency (AMD), morphological abnormalities were confined to skeletal muscle and consisted of a vacuolar myopathy. Acid maltase activity, however, was approximately 6% of normal in muscle, liver, and brain, and 3% of normal in heart. Kinetic characteristics, and inhibition by antibodies and Zn++, showed that the residual activity was “authentic” acid maltase. Neutral maltase activity was normal in muscle and liver, but decreased in brain (55% of normal) and heart (19% of normal). Although the relative decrease of acid maltase was similar in different tissues, absolute residual activity was lowest in skeletal muscle: this may explain the selective involvement of this tissue in late-onset AMD.     

A freeze-fracture analysis of intramembrane particle densities on dystrophic hamster heart sarcolemma

The intramembrane particle (IMP) profile of control and dystrophic (Bio 14.6) hamster cardiac muscle plasma membrane was assessed in freeze-fracture replicas to determine whether this animal model of muscular dystrophy exhibits the same membrane characteristics found in skeletal muscle from other more thoroughly studied dystrophic animals, and to test the hypothesis that the plasma membrane of the cardiac muscle cell is the site of a defect associated with the disease. Samples of cardiac muscle tissue from hamsters ranging in age from 1 to 13 months were freeze-fractured. Intramembrane particle numbers were determined for all tissue samples by counting randomly selected areas of P- and E-face surfaces. Up to the age of 1 month, the particle density was the same in both strains of hamster, after which time, the population of IMPs was about 30% lower in dystrophic than in normal heart sarcolemma. This 30% difference in particle frequency in dystrophic hamster heart membrane is consistent with values published for cell membrane from other muscular dystrophies and supports the theory that there is a defect in the plasma membrane of dystrophic cells. In addition, this study has shown for the first time that a presumed membrane defect related to muscular dystrophy (reduced number of IMPs) may be present throughout the life of the animal (1-13 months), and expressed in every cell sampled.