Bioehimica et Biophysica Acta, 400 (1975) 255-257
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands BBA 37120 STRUCTURAL DETERIORATION OF TENDON COLLAGEN IN GENETIC MUSCULAR DYSTROPHY
ROBERT H. STINSON Department of Physics, University of Guelph, Guelph, Ontario (Canada)
(Received February 10th, 1975)
SUMMARY The structure of gastrocnemius tendons from chickens with genetically induced muscular dystrophy has been studied by low-angle X-ray diffraction. Compared with normal samples there is poor alignment of collagen within the tendons. This difference is quite pronounced at eight weeks when the affected birds are still in comparatively good physical condition. Similar changes have been reported for birds with nutritionally induced muscular dystrophy (Bartlett, M. W., Egelstaff, P. A., Holden, T. M., Stinson, R. H. and Sweeny, P. R. (1973) Biochim. Biophys. Acta 328, 213-220).
INTRODUCTION Muscle and associated nerve tissue has been extensively investigated in the search for a greater understanding of the aetiology of muscular dystrophy. Marked deterioration of the muscles is the most obvious symptom of the disease but is by no means the only symptom. Deterioration of connective tissue structure has been shown to occur at early stages in the development of muscular dystrophy [1--4]. It has been reported [1] that the diffraction pattern obtained from collagen in intact gastrocnemius tendons obtained from 18-day ducklings rendered dystrophic nutritionally was much weaker in intensity than that for normal tendon. The intensities of various orders were four times greater for the healthy birds than for the sick. The disorientation of the collagen was measured as 12° for the normal and 36 ° for the dystrophic. At this age the sick birds were in very poor condition and unable to walk. It seemed reasonable to ask if similar changes occur in genetically induced muscular dystrophy. This report provides an affirmative answer for the changes that can be revealed by low-angle X-ray diffraction. EXPERIMENTAL A control line (200) of New Hampshire chickens as well as lines with muscular dystrophy of genetic origin (304, 307 and 308) were obtained from Dr D. W. Peterson, Dept. of Avian Sciences, University of California, Davis, Calif., U.S.A. The birds
256 were raised under identical conditions, killed at eight weeks from hatching and gastrocnemius tendons removed for investigation. The tendons in a wet state were examined with a Rigaku-Denki small angle X-ray diffractometer. A scanning proportional counter was used as a detector. The geometry of the data collection system was such as to integrate over any subsidiary offmeridional maxima and also over any arcing due to moderate disorientation within the samples. The complete system and methods of data reduction have been fully described [1 ]. The "integrated" intensities do not correspond to the usual crystallographic definition of the term because the effect of mosaic spread has not been allowed for. As will be shown later the "mosaic spread" is really a disorientation of collagen within the tendon and is too large to permit the usual technique of rotating the sample in company with the detector. This disorientation was measured with 2 0 set at the peak intensity of the third order. In this position the incident X-ray beam and the fibre axis of the tendon were mutually perpendicular and horizontal. The tendon was then rotated about the vertical axis through the point of intersection of the beam and the sample. The disorientation given is the total angular range within which the intensity of the diffracted beam remains at least half that found when the sample is normal to the beam. Only third orders were used as a compromise between the mutually conflicting requirements for high peak intensity and low, linear background. Orders higher than the third had low peak intensities, especially in the dystrophic samples, while the background around the first order is apparently non-linear. RESULTS AND DISCUSSION When sacrificed the control line was able to pass the "flip test" but the others could not, i.e. the dystrophic birds could not fight themselves when placed on their backs. At this age the dystrophic birds could not be identified by casual observation and appeared to be walking normally. Closer investigation revealed that, in addition to failure to pass the flip test, there was considerable characteristic deterioration of breast muscle tissue with higher than normal amounts of fatty tissue. Thus, the disease had not progressed nearly as far in these dystrophic birds as in the nutritionally deprived birds in the earlier report. The relative intensities of the various orders within a diffraction pattern were essentially constant within both the control and diseased groups indicating no significant change in the distribution of electron density within individual unit cells in disease. The ratio of the intensity of the third reflection to the fifth for eight randomly selected samples from all strains was 2.69 with a standard deviation of 0.18, a value well within the limits imposed by measurement uncertainties. The length of the unit cell was quite uniform. For the same eight samples the average length was 678 ± 3/~. The absolute intensities of the reflections were always much greater for the control strain than for any of the diseased strains with the average intensity for the diseased strains being approximately one-half that of the normal, for any given order. There was no significant difference within the diseased strains. The disorientation of the control line was found to be 8.7 ° while the average and standard deviation for the three affected strains was 12.9 ~ 1.6 °. This was meas-
257 ured for the third orders only, not only because of their greater intensity but also because it has long been realized [5] that the spreading out of reflections over layer lines was roughly proportional to the index of the diffraction order. The geometry of the detection system was such as to integrate over any "arcing" of reflections brought about by disorientations of the magnitude encountered. In the earlier report [1 ] the sick birds from which the samples were taken had deteriorated to the point where they were unable to walk. Disuse might then have been a factor in the increased disorientation of collagen within the tendon. The birds in this present investigation were still walking well and the increased disorder of tendon elements is attributed to the existence of muscular dystrophy of genetic origin rather than to disuse. ACKNOWLEDGEMENTS The financial assistance of the National Research Council of Canada is gratefully acknowledged. The author is indebted to Mr Wilson Ayoung-Chee for technical assistance. REFERENCES 1 Bartlett, M. W., Egelstaff, P. A., Holden, T. M., Stinson, R. H. and Sweeny, P. R. (1973) Biochim. Biophys. Acta 328, 213-220 2 Sweeny, P. R. and Brown, R. G. (1972) Am. J. Pathol. 68, 479-486 3 Sweeny, P. R., Buchannan-Smith, J. G., de Mille, F., Pettit, J. R. and Moran, E. T. (1972) Am. J. Pathol. 68, 493-504 4 Bourne, G. H. and Golarz, M. N. (1963) J. Histochem. Cytochem. 11,286-288 5 Bear, R. S. (1952) in Advances in Protein Chemistry (Anson, M. L., Bailey, K. and Edsall, J. T., eds), Vol. 7, p. 69, Academic Press, New York
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands BBA 37120 STRUCTURAL DETERIORATION OF TENDON COLLAGEN IN GENETIC MUSCULAR DYSTROPHY
ROBERT H. STINSON Department of Physics, University of Guelph, Guelph, Ontario (Canada)
(Received February 10th, 1975)
SUMMARY The structure of gastrocnemius tendons from chickens with genetically induced muscular dystrophy has been studied by low-angle X-ray diffraction. Compared with normal samples there is poor alignment of collagen within the tendons. This difference is quite pronounced at eight weeks when the affected birds are still in comparatively good physical condition. Similar changes have been reported for birds with nutritionally induced muscular dystrophy (Bartlett, M. W., Egelstaff, P. A., Holden, T. M., Stinson, R. H. and Sweeny, P. R. (1973) Biochim. Biophys. Acta 328, 213-220).
INTRODUCTION Muscle and associated nerve tissue has been extensively investigated in the search for a greater understanding of the aetiology of muscular dystrophy. Marked deterioration of the muscles is the most obvious symptom of the disease but is by no means the only symptom. Deterioration of connective tissue structure has been shown to occur at early stages in the development of muscular dystrophy [1--4]. It has been reported [1] that the diffraction pattern obtained from collagen in intact gastrocnemius tendons obtained from 18-day ducklings rendered dystrophic nutritionally was much weaker in intensity than that for normal tendon. The intensities of various orders were four times greater for the healthy birds than for the sick. The disorientation of the collagen was measured as 12° for the normal and 36 ° for the dystrophic. At this age the sick birds were in very poor condition and unable to walk. It seemed reasonable to ask if similar changes occur in genetically induced muscular dystrophy. This report provides an affirmative answer for the changes that can be revealed by low-angle X-ray diffraction. EXPERIMENTAL A control line (200) of New Hampshire chickens as well as lines with muscular dystrophy of genetic origin (304, 307 and 308) were obtained from Dr D. W. Peterson, Dept. of Avian Sciences, University of California, Davis, Calif., U.S.A. The birds
256 were raised under identical conditions, killed at eight weeks from hatching and gastrocnemius tendons removed for investigation. The tendons in a wet state were examined with a Rigaku-Denki small angle X-ray diffractometer. A scanning proportional counter was used as a detector. The geometry of the data collection system was such as to integrate over any subsidiary offmeridional maxima and also over any arcing due to moderate disorientation within the samples. The complete system and methods of data reduction have been fully described [1 ]. The "integrated" intensities do not correspond to the usual crystallographic definition of the term because the effect of mosaic spread has not been allowed for. As will be shown later the "mosaic spread" is really a disorientation of collagen within the tendon and is too large to permit the usual technique of rotating the sample in company with the detector. This disorientation was measured with 2 0 set at the peak intensity of the third order. In this position the incident X-ray beam and the fibre axis of the tendon were mutually perpendicular and horizontal. The tendon was then rotated about the vertical axis through the point of intersection of the beam and the sample. The disorientation given is the total angular range within which the intensity of the diffracted beam remains at least half that found when the sample is normal to the beam. Only third orders were used as a compromise between the mutually conflicting requirements for high peak intensity and low, linear background. Orders higher than the third had low peak intensities, especially in the dystrophic samples, while the background around the first order is apparently non-linear. RESULTS AND DISCUSSION When sacrificed the control line was able to pass the "flip test" but the others could not, i.e. the dystrophic birds could not fight themselves when placed on their backs. At this age the dystrophic birds could not be identified by casual observation and appeared to be walking normally. Closer investigation revealed that, in addition to failure to pass the flip test, there was considerable characteristic deterioration of breast muscle tissue with higher than normal amounts of fatty tissue. Thus, the disease had not progressed nearly as far in these dystrophic birds as in the nutritionally deprived birds in the earlier report. The relative intensities of the various orders within a diffraction pattern were essentially constant within both the control and diseased groups indicating no significant change in the distribution of electron density within individual unit cells in disease. The ratio of the intensity of the third reflection to the fifth for eight randomly selected samples from all strains was 2.69 with a standard deviation of 0.18, a value well within the limits imposed by measurement uncertainties. The length of the unit cell was quite uniform. For the same eight samples the average length was 678 ± 3/~. The absolute intensities of the reflections were always much greater for the control strain than for any of the diseased strains with the average intensity for the diseased strains being approximately one-half that of the normal, for any given order. There was no significant difference within the diseased strains. The disorientation of the control line was found to be 8.7 ° while the average and standard deviation for the three affected strains was 12.9 ~ 1.6 °. This was meas-
257 ured for the third orders only, not only because of their greater intensity but also because it has long been realized [5] that the spreading out of reflections over layer lines was roughly proportional to the index of the diffraction order. The geometry of the detection system was such as to integrate over any "arcing" of reflections brought about by disorientations of the magnitude encountered. In the earlier report [1 ] the sick birds from which the samples were taken had deteriorated to the point where they were unable to walk. Disuse might then have been a factor in the increased disorientation of collagen within the tendon. The birds in this present investigation were still walking well and the increased disorder of tendon elements is attributed to the existence of muscular dystrophy of genetic origin rather than to disuse. ACKNOWLEDGEMENTS The financial assistance of the National Research Council of Canada is gratefully acknowledged. The author is indebted to Mr Wilson Ayoung-Chee for technical assistance. REFERENCES 1 Bartlett, M. W., Egelstaff, P. A., Holden, T. M., Stinson, R. H. and Sweeny, P. R. (1973) Biochim. Biophys. Acta 328, 213-220 2 Sweeny, P. R. and Brown, R. G. (1972) Am. J. Pathol. 68, 479-486 3 Sweeny, P. R., Buchannan-Smith, J. G., de Mille, F., Pettit, J. R. and Moran, E. T. (1972) Am. J. Pathol. 68, 493-504 4 Bourne, G. H. and Golarz, M. N. (1963) J. Histochem. Cytochem. 11,286-288 5 Bear, R. S. (1952) in Advances in Protein Chemistry (Anson, M. L., Bailey, K. and Edsall, J. T., eds), Vol. 7, p. 69, Academic Press, New York
