Small Angle X-ray Diffraction as a Tool for Structural Characterization of Muscle Disease

Abstract

Small angle X-ray fiber diffraction is the method of choice for obtaining molecular level structural information from striated muscle fibers under hydrated physiological conditions. For many decades this technique had been used primarily for investigating basic biophysical questions regarding muscle contraction and regulation and its use confined to a relatively small group of expert practitioners. Over the last 20 years, however, X-ray diffraction has emerged as an important tool for investigating the structural consequences of cardiac and skeletal myopathies. In this review we show how simple and straightforward measurements, accessible to non-experts, can be used to extract biophysical parameters that can help explain and characterize the physiology and pathology of a given experimental system. We provide a comprehensive guide to the range of the kinds of measurements that can be made and illustrate how they have been used to provide insights into the structural basis of pathology in a comprehensive review of the literature. We also show how these kinds of measurements can inform current controversies and indicate some future directions.

Keywords: X-ray diffraction; cardiomyopathy; muscle; myopathy.

Figure 1

X-ray diffraction patterns. (A) X-ray diffraction patterns from intact mouse EDL muscle during resting (left panel) and contracting (right panel) conditions (based on reference [24]). (B) X-ray diffraction patterns from permeabilized porcine cardiac tissue at pCa8 (left panel) and pCa5.8 (right panel) (based on reference [49]). Panels A and B are on approximately the same intensity scale. The signal to noise is much better in the patterns shown in panel A since they are from whole intact skeletal muscle and the patterns shown in panel B are from a small cardiac fiber bundle.

Figure 2

Equatorial reflections from muscle X-ray diffraction patterns (A) Cross-sectional view of sarcomere A band. The thick filaments (thick dots) form a 2D hexagonal lattice and the thin filaments (thin dots) interdigitate into the trigonal points between the thick filaments. (B) Equatorial reflections (up panel) and their one-dimensional intensity profiles (bottom panel) from permeabilized murine myocardium under resting (pCa 8) and sub-maximally activated state (pCa5.8) (based on reference [50]).

Figure 3

Electron density maps. Axially projected electron density maps calculated from the first five equatorial reflections. A: thin filament; M: thick filament. Calibration bar = 50 nm. Reprinted from Ref. [75]. In the difference maps, colored regions are those that have increased density at longer sarcomere length with brighter colors indicating greater increased density.

Figure 4

The origin of meridional and layer line reflections. Schematic diagram of the quasi-helical arrangement of myosin heads around the thick filament backbone (left panel) and the double helical arrangement of actin monomers in the thin filament (right panel). The meridional reflections arising from these features are shown in the composite X-ray diffraction pattern (based on reference [24]) in the center panel with the left half from relaxed muscle and the right from contracting mouse soleus muscle. Note that the M6 and higher order myosin meridional reflections come from structures within the thick filament backbone (not shown on the figure).

Figure 5

The M1 and M2 clusters in the meridional pattern (see reference [75]). The M1 and M2 clusters are located around the expected positions of the M1 and M2 “forbidden” meridional reflections. C_1,1, C_1.2 and C_2,1, C_2,2 are doublets assumed to correspond the first order and second order MyBP-C reflections, respectively. Tn1 and Tn2 are the first and second order troponin meridional reflections, respectively. In this example, the M1 forbidden myosin reflection is not resolved and only the M2 is visible.

Figure 6

Relationship between the radius of the helical diffracting object and the distribution of intensity on a layer line. R_m is the radius to the center of mass of the quasi-helically arranged myosin heads and R_A is the radius to the center of mass of an actin subunit. J₀–J₃ (generally J_n where n is an integer) are Bessel functions of the first kind. The order of the Bessel function (n) can be determined from Equation (1) or Equation (2).