Myofibril orientation as a metric for characterizing heart disease

Myocyte disarray is a hallmark of cardiomyopathy. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle sigma). At a sarcomere length (SL) of 1.9 microns, the angle sigma was 0.23 +/- 0.01 rad, decreased to 0.19 +/- 0.01 rad at a SL of 2.1 microns, and further decreased to 0.15 +/- 0.01 rad at a SL of 2.3 microns (p<0.0001). Angle sigma was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24 +/- 0.01 rad) compared to WT myocardium (0.14 +/- 0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19 +/- 0.006 rad) when compared to non-failing samples (0.17 +/- 0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological methods that directly assess myocyte orientation and only indirectly assess myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating the structural bases of cardiomyopathies.

Abstract P350: Myofibril Orientation In Cardiac Muscle And Its Implication For Heart Disease

Rationale: Myocyte disarray is a hallmark of cardiomyopathy. However, the orientation of individual myofibrils and myofilaments and how their alignment may be altered in disease progression have been largely underexplored. This oversight has been predominantly due to a paucity of methods for objective and quantitative analysis. Objective: To introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority versus traditional histological assessments. Methods and Results: Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in skinned, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length (SL) of 1.9 μm, the angle σ was 0.23±0.01 rad, decreased to 0.19±0.01 rad at a SL of 2.1 μm, and further decreased to 0.15±0.01 rad at a SL of 2.3 μm (p<0.0001). Angle σ was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24±0.01 rad) compared to WT myocardium (0.14±0.005 rad, p<0.0001) as well as in biopsied human heart failure tissue (0.19±0.006 rad) when compared to non-failing samples (0.17±0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from myofibrillar disorientation compared to healthy controls. Finally, using control samples, we showed that traditional, histological-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Conclusions: Our method for assessing myofibrillar orientation limits the artifacts introduced by traditional histological processing and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen, biopsied human myocardium opens up new avenues of inquiry regarding the relation of myofibrillar structure to function in health and disease.

A wide-angle X-ray fibre diffraction method for quantifying collagen orientation across large tissue areas: application to the human eyeball coat

A quantitative map of collagen fibril orientation across the human eyeball coat, including both the cornea and the sclera, has been obtained using a combination of synchrotron wide-angle X-ray scattering (WAXS) and three-dimensional point mapping. A macromolecular crystallography beamline, in a custom-modified fibre diffraction setup, was used to record the 1.6 nm intermolecular equatorial reflection from fibrillar collagen at 0.5 mm spatial resolution across a flat-mounted human eyeball coat. Fibril orientation, derived as an average measure of the tissue thickness, was quantified by extraction of the azimuthal distribution of WAXS scatter intensity. Vector plots of preferential fibre orientation were remapped onto an idealized eyeball surface using a custom-built numerical algorithm, to obtain a three-dimensional representation of the collagen fibril architecture.

DEVELOPMENT OF FEATHER KERATIN DURING EMBRYOGENESIS OF THE CHICK

The development of keratin in 9 to 17 day embryonic chick feathers has been studied by x-ray diffraction and cytochemical methods. The x-ray diffraction pattern given by the 9-day feathers contains none of the features seen in the adult pattern. In the 10 to 11 day patterns, besides two diffuse rings centered at 4.7 A and 10 A, two sharp, rather weak rings are seen at 35 A and 4.2 A with slight preferred orientations about the equator and the meridian, respectively. At 12 days, in addition to the foregoing, a sharp intense equatorial reflection at ∼56 A is observed. On treatment with lipid solvents, the 35 A ring is removed; prolonged extraction removes the 4.2 A ring, while blurring the 56 A reflection and enhancing the central low angle scatter. The 14-day pattern shows, besides all the features seen in the earlier patterns, a 23 A meridional reflection and other meridional and near meridional reflections. All the basic features of the adult pattern are seen at this stage and remain essentially intact on lipid extraction. Beyond 14 days, the pattern remains essentially the same, only improving in clarity and detail. The 4.2 A ring seen in the 10 to 15 day pattern is scarcely detectable in the 16-day pattern. Cytochemical evidence indicates that extensive —S—S bond formation occurs between the 13th and 14th days. It is suggested that lipids serve as a framework for the developing keratin structure which acquires permanent stability through hydrogen bonds and disulfide cross-links. The relation between keratin synthesis and tissue architecture as well as cytodifferentiation is discussed.