Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs

Abstract

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles.

Fig 1. Actin orientation guided segmentation of the α-actinin skeleton.

A, Images of cardiac (Ai) and skeletal muscle (Aii) stained for actin fibrils (green), α-actinin (red), and nuclei (blue). In Ai, off-target α-actinin stain is outlined in white and the region containing z-lines is outlined with a yellow dashed-dotted line. B, The orientation of the actin fibrils in (A), represented by green arrows, plotted on top of the α-actinin skeleton. C, Actin orientation vectors (green) overlaid on α-actinin stained cardiac tissue (Ai) where each pixel in the α-actinin binary skeleton (Bi) is colored according to its orientation relative to local actin orientation from parallel (dark blue) to perpendicular (red) as indicated by the colorbar. D, Skeletal muscle shown in Aii with off-target α-actinin staining (blue) and z-lines (red). Scale bars: 15 μm.

Fig 2. Automatic detection of continuous z-line lengths.

Perfectly (A) and variably (B) continuous synthetic data. Ai and Bi, Synthetic data composed of four registered and continuous segments that were each seven pixels long. Segments that were shifted over by one pixel were treated as continuous. Aii and Bii, Continuous lines plotted as distinct colors on top of the synthetic data. C, The length of each continuous line detected in Aii and Bii, with the colors corresponding to those in Aii and Bii. The dashed lines indicate the number of pixels that composed continuous segments.

Fig 3. Analysis of cardiomyocytes with variable aspect ratios.

A, (top) Images of single cells with area 2500 μm², but variable aspect ratios ((Ai) 1:1, (Aii) 3:1 (Aiii) 6:1 (Aiv) 11:1) stained for actin (green) and α-actinin (red) and their corresponding continuous z-lines (bottom). B, Representative α-actinin stained cardiomyocytes (extracellular matrix (ECM) island ~6:1 aspect ratio) for each z-line architecture classification: (Bi) good z-line architecture, (Bii) good z-line architecture with bad spread in ECM island, (Biii) intermediate z-line architecture, and (Biv) immature, underdeveloped (bad) z-line architecture. C, Average z-line fraction for cells. D, Average median continuous z-line length for cells (n = 101) within each classification of z-line architecture as described in C. E, Mean and standard deviation of the number of z-line pixels for the good cells of each aspect ratio. F, Mean and standard deviation of the estimated force for the good cells of each aspect ratio. Groups were compared using ANOVA with Tukey’s test p <0.05 (black bars in C, E, and F). Scale bar: 15 μm.

Fig 4. Comparison of cardiac tissues.

A-D, Cardiac tissue stained for actin (green), α-actinin (red), and nuclei (blue) on a uniform layer of FN (A), FN in lines (B), FN in lines with sparsely seeded cardiomyocytes (C), and FN in lines with cardiomyocytes treated with BDM (D). E, Z-line OOP. F, Z-line fractions. G, Median continuous z-line lengths. H, Nuclei per area. In E-H Each dot represents a coverslip. In E-H, each dot represents a single coverslip, colored bars represent the mean, and colored boxes represent the standard deviation. Groups were compared using ANOVA with Tukey’s test p <0.05 (black bars in E, F, G and H). Number of coverslips (cs) for each condition: isotropic cs = 12, anisotropic cs = 11, sparse anisotropic cs = 6, BDM treated cs = 5. Scale bars: (A-Di) 50 μm; (A-D ii) 15 μm.