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 σ). At a sarcomere length of 1.9 μm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 μm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 μm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 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.

Figure 1

Equatorial x-ray diffraction patterns from permeabilized murine myocardium. Representative equatorial x-ray diffraction patterns from permeabilized mouse myocardium (A–C) and the angular intensity profile of the 1,0 equatorial reflection (D) at different sarcomere lengths. The full width at half maximum (∼2.36 σ) of the peaks is indicated by the double-headed arrows at corresponding colors.

Figure 2

1,0 equatorial reflections in permeabilized mouse myocardium at different sarcomere lengths. (A) The angular standard deviation of 1,0 equatorial reflections (angle σ) from permeabilized mouse myocardium as a function of sarcomere length. (B) The standard deviation of the 1,0 equatorial reflections in radial direction (width σ) from permeabilized mouse myocardium as a function of sarcomere length (^nsp ≥ 0.05, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001).

Figure 3

Myofibrillar orientation from permeabilized porcine myocardium. (A) Representative equatorial x-ray diffraction patterns from permeabilized WT and R403Q porcine myocardium. (B) The angular standard deviation of 1,0 equatorial reflections (angle σ) from permeabilized WT and R403Q porcine myocardium. (C) The standard deviation of the 1,0 equatorial reflections in radial direction (width σ) from permeabilized WT and R403Q porcine myocardium (^∗∗∗∗p < 0.0001).

Figure 4

Representative x-ray patterns from frozen Non-Failing (A) and patients with HF (B) human myocardium. The 1,0 and 1,1 equatorial reflections, the third (M3) and sixth (M6) order myosin-based meridional reflections, first-order myosin-based layer lines (MLL1), and sixth order actin-based layer lines (ALL6) are as labeled.

Figure 5

Human myocardium myofibrillar orientation by x-ray from Non-Failing and patients with HF. (A) Representative equatorial x-ray diffraction patterns from Non-Failing myocardium and HF myocardium. (B) The angular standard deviation of 1,0 equatorial reflections (angle σ) from permeabilized Non-Failing and HF human myocardium. (C) The standard deviation of the 1,0 equatorial reflections in radial direction (width σ) from permeabilized Non-Failing and HF human myocardium (^∗p < 0.05).

Figure 6

Human cardiomyocyte alignment by histology analysis from Non-Failing and patients with HF (HFrEF). Representative histology images from Non-Failing myocardium (A) and HF myocardium (B). (C) Representative histology images from Non-Failing myocardium at an alternative region. (D) The percentage of aligned myocytes in Non-Failing myocardium, HFrEF myocardium, and Non-Failing myocardium at alternative regions (Non-Failing Alt) that were not considered longitudinally aligned or suitable for analysis from histology analysis (^nsp ≥ 0.05, ^∗∗∗∗p < 0.0001).