Myofibril orientation as a metric for characterizing heart disease

Abstract

Myocyte disarray is a hallmark of many cardiac disorders. 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 because of 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 with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle s). At a sarcomere length of 1.9 mm, the angle s was 0.23 5 0.01 rad, decreased to 0.19 5 0.01 rad at a sarcomere length of 2.1 mm, and further decreased to 0.15 5 0.01 rad at a sarcomere length of 2.3 mm (p < 0.0001). Angle s was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 5 0.01 rad) compared with wild-type myocardium (0.14 5 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 5 0.006 rad) when compared with nonfailing samples (0.17 5 0.007 rad; p ¼ 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with 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 approaches that assess myocyte orientation and only indirectly evaluate 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 structural anomalies associated with cardiac diseases.

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

“…Transgenic rat and porcine models of myopathies are relatively uncommon but are becoming increasingly available. There have been relatively few studies on human skeletal [ 37 , 39 , 173 ] and cardiac muscle [ 40 ], but the early indications are that the quality of diffraction patterns from human muscle are at least as high as from porcine myocardium, indicating that diffraction techniques can be applied to questions of direct clinical relevance.…”

“…An exciting new focus, with only a relatively small number of studies so far, e.g., [ 30 , 35 , 36 , 37 ], is on X-ray diffraction of human skeletal and cardiac muscle. Human skeletal and cardiac muscle can yield detailed diffraction patterns suggesting that studies of muscle from human biopsies can be particularly valuable to study structural phenotypes of many muscle diseases, although only a few reports have appeared so far e.g., [ 36 , 37 , 38 , 39 , 40 ]. Finally, X-ray diffraction is beginning to be used to explore the mechanism of action of experimental drugs [ 41 , 42 ] on normal and transgenic porcine myocardium that is emerging as a much better model of human hearts than rodent systems.…”

“…In striated muscle, the equatorial reflections are perpendicular to the myofibrillar longitudinal axis; thus, the angular spread, angle σ, of the equatorial reflections across the equator ( Figure 1 ) can be used to measure the degree of alignment of the sarcomeres in the myofibrils relative to the long axis of the preparation [ 40 ]. Angle σ can be easily estimated from equatorial patterns using the Diffraction Imaging tool in MuscleX.…”

“…Angle σ can be easily estimated from equatorial patterns using the Diffraction Imaging tool in MuscleX. Angle σ has been shown to decrease with increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium [ 40 ].…”

“…Note that conventional histological approaches [ 84 ] measure cellular level (myocyte) orientation rather than myofibrillar/myofilament level orientation. Using angle σ from the equatorial diffraction pattern as an estimate of myofibrillar orientation avoids the artifacts introduced by conventional histological processing and provides a precise and objective metric for phenotypically characterizing myocardium [ 40 ]. This new metric may be expected to provide a generally applicable tool for establishing the relationship of myofibrillar (as opposed to the cellular level) orientation to muscle performance in future studies.…”