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.

Cardiac myosin filaments are directly regulated by calcium

“…Since I M6 arises primarily from the thick filament backbone, the decrease of I M6 in the presence of Ca 2+ suggests a structural change in the thick filament backbone induced by Ca 2+ , independent of thick filament strain. The spacing of the M6 reflection (S M6 ), which reports the periodicity of the thick filament backbone ( Ma and Irving, 2022 ), increases to 0.73 ± 0.06% at the fully activated state (pCa 4.5) in the control group and 0.36 ± 0.02% in the inhibitor group where active force is absent ( Fig. 3 d and Table S2 ).…”

“…In a resting muscle, most myosin heads are quasi-helically ordered on the surface of the thick filament, where these off -state myosin heads produce the myosin-based layer line reflections. Myosin heads lose their helical order when turned on to participate in contraction ( Huxley and Brown, 1967 ; Ma and Irving, 2022 ). The intensity of the first-order myosin-based layer line (I MLL1 ) and the third-order myosin-based meridional reflection (I M3 ), both of which correlate with the ordering of myosin heads ( Reconditi, 2006 ; Ma and Irving, 2022 ), decreases progressively in the presence of increasing Ca 2+ .…”

“…Myosin heads lose their helical order when turned on to participate in contraction ( Huxley and Brown, 1967 ; Ma and Irving, 2022 ). The intensity of the first-order myosin-based layer line (I MLL1 ) and the third-order myosin-based meridional reflection (I M3 ), both of which correlate with the ordering of myosin heads ( Reconditi, 2006 ; Ma and Irving, 2022 ), decreases progressively in the presence of increasing Ca 2+ . The I MLL1 reflection changes ( Fig.…”

“…The spacings of targeted reflections are estimated by measuring the distance from the beam center to the peak position as the centroid of the intensity in profile, considering only the top half of the diffraction peak ( Huxley et al, 1994 ; Kiss et al, 2018 ). The center of mass of the cross-bridges (R m ) is estimated based on modeling the layer line as a J3 Bessel function with the argument 4.2 = 2 × π × r × R m , where r is a reciprocal space coordinate as described ( Ma et al, 2021 ; Ma and Irving, 2022 ). To compare the intensities under different conditions, the measured intensities of x-ray reflections are normalized to the sixth-order actin-based layer line intensities.…”

MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips

“…We studied the X-ray diffraction patterns from relaxed permeabilized porcine myocardium at different [ADP]. Qualitatively, permeabilized porcine myocardium showed characteristic relaxed myosin based layer lines (MLL1 and MLL2) originating from the quasi-helically ordered arrangement of resting myosin heads [ 30 ] in the absence of ADP ( Figure 1 ). The intensities of these reflections decreased as [ADP] increased, indicating a progressive loss of the helical ordering of the myosin heads.…”