Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte

Figures

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  Figure 1.. Healthy cardiomyocytes exhibit nonuniform sarcomere strain. A, Sarcomeres in isolated cardiomyocytes were tracked by CellMask labeling of t tubules. High-speed confocal imaging of 2-dimensional regions was performed during 1 Hz field stimulation at 37 °C (scale bar, 5 μm). Parabolic fitting was used to identify peak transverse signals, with each peak defining a Z-line position (original data, gray; fitted curve, red; fitted peak, dark blue; see also Figure S1). By subtracting neighboring Z-line positions, sarcomere length (SL) change traces were plotted. During cell contraction, most sarcomeres shortened normally (dark blue traces in bottom), but some sarcomeres were lengthened (orange traces). B, Sarcomere deformations from neighboring sarcomeres are plotted in the same graphs, from 2 regions indicated in A (dashed gray frames). C, Proportions of sarcomeres exhibiting shortening, stretch, or no change in length. D, Resting SLs were observed to be nonhomogeneous, and stretched sarcomeres were significantly shorter than those that shortened. Data are presented as mean±SEM together with raw data. nsarcomeres: shortening, 130; stretch, 15; stationary, 7 from 13 cells, 5 hearts.…

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  Figure 2.. Differential sarcomere deformation occurs despite uniform Ca2+ transients. A, Epifluorescent microscopy was used to simultaneously monitor cytosolic Ca2+ and sarcomere striations, using fluo-4 fluorescence and bright-field signals, respectively (see also Figure S2). Bright-field images (left) were filtered with a fast Fourier transform and inverted to assign Z-line signals the highest values (orange). Parabolic fitting was performed to identify peak transverse signals, defining Z-line positions (original data, gray; fitted curve, red; fitted peak, dark blue). By averaging Z-line movement from 5 consecutive contractions, sarcomere deformation traces were plotted. Z-line positions were also used to define local Ca2+ transients for each sarcomere (right; fluorescence at peak of the Ca2+ transient is illustrated at the top). Despite uniform Ca2+ transients observed at individual sarcomeres, differential sarcomere strain was observed (bottom). Scale bars, 5 μm. B, Local Ca2+ transients and deformations from the neighboring sarcomeres indicated in A (dashed gray frames) are plotted in the same graph. C, Proportions of sarcomeres exhibiting shortening, stretch, or no change in length,…

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  Figure 3.. Length-dependent activation in single cardiomyocytes is associated with harmonization of sarcomere strain. A, Cardiomyocytes were attached to glass fibers and stretched in intervals corresponding to a 5% command increase in length. Developed force recordings are presented for the illustrated cardiomyocyte, with data (mean±SEM) presented at right (sigmoidal curve fitting; see Figure S4 for raw recording). B, Sarcomere deformations in a representative cardiomyocyte at 0% and 20% stretch. C, Local Ca2+ transients and sarcomere deformations from the neighboring sarcomeres indicated in B. D, Progressive stretch increased the proportion of sarcomeres exhibiting shortening. E, During cell stretch, resting sarcomere length remained longer in shortening than stretched sarcomeres. F, Increasing magnitude sarcomere shortening during cell lengthening. Data in D through F are presented as mean±SEM with regression line. The same sarcomeres were examined in all panels. nsarcomeres: 0% stretch, 86; 5% stretch, 100; 10% stretch, 100; 15% stretch, 109; 20% stretch, 121, from 6 cells, 6 hearts. Statistical methods: (A) 1-way repeated measures ANOVA to test the hypothesis that cell stretch increased…

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  Figure 4.. Mathematical modeling of sarcomere recruitment during length-dependent activation. A, Mathematical modeling of an array of 15 sarcomeres arranged in 3 myofibrils, with parallel connections via elastic springs at Z lines. Sarcomere length (SL) was varied for a central test sarcomere, while the length of other sarcomeres was maintained at 1.75 µm (the averaged resting SL in experimental data). B, Sarcomeres were stimulated by a representative experimental Ca2+ transient. C, The steady-state force-length dependence derived from Rice et al,30 quantifying the region where a single thin filament overlaps with the crossbridge containing region of the thick filament. D and E, Modeled strain of test sarcomere in A as a function of resting SL. F, An expanded array of 150 sarcomeres arranged in 30 myofibrils was used to include the SL variability observed experimentally. The SLs were randomly generated by using a normal distribution with mean of 1.75 µm and 15% SD (Figure 3D; 0% stretch). Proportions of shortening, stretched, and stationary sarcomeres. G, Distribution of resting SL values for each type of strain. H, Stepwise lengthening of the sarcomere array progressively increased the…

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  Figure 5.. Cell lengthening harmonizes sarcomere strain patterns, augmenting work performance. Schematic overview of findings, showing the presence of both shortening and stretched sarcomeres within myofibrils. Stretched sarcomeres are illustrated with shorter sarcomere length. Lower likelihood of crossbridge formation at these sarcomeres may reflect larger interfilament lattice spacing (illustrated) or lower stretch-dependent activation of the thin and thick filaments. Progressive stretch of the myofibril increases the fraction of shortening sarcomeres, as lengthening the shortest sarcomeres recruits their active force generation (to overcome the stretching effects of their longer neighbors). Increasing sarcomere recruitment during cell lengthening augments overall contractile efficiency, as less wasted (negative) work is performed.

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  Figure 6.. Titin regulates sarcomere dimensions and intersarcomere dynamics. A, Representative micrographs from fixed WT (wild type) and Ttntv/+ mouse myocardium. Enlargements of 2 neighboring sarcomeres indicate the length of the I band (red bar), A band (green), and overall sarcomere length (blue) and highlight the variability of these dimensions. Scale bars, 0.2 μm. B, Mean I band, A band, and total sarcomere lengths (±SEM). C, Variability in sarcomere dimensions, calculated as SD within each image, presented as mean±SEM. nsarcomeres: WT, 71 from 3 hearts; Ttntv/+, 72 from 3 hearts. D, Sarcomere tracking experiments in Ttntv/+ cells revealed fewer shortening and more stretched sarcomeres than in WT (dark blue traces, shortening; orange traces, stretched). E, Proportions of shortening, stretched, and stationary sarcomeres. nsarcomeres: WT, 260 from 23 cells, 3 hearts; Ttntv/+, 248 from 19 cells, 3 hearts. Statistical methods: (B and C) Mann-Whitney U test; (E) χ2 test (proportion stretched sarcomeres in WT vs TTNtv/+, P=0.00396). P<0.05 was considered significant (*P<0.05, ***P<0.001). Detailed statistical results are shown in Table S9.

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  Figure 7.. During lengthening, Ttntv/+ cardiomyocytes exhibit diminished length-dependent activation and passive stiffness and greater nonuniformity of sarcomere strain. A, Representative developed force recordings for WT (wild type) and Ttntv/+ cardiomyocytes during stepwise stretch experiments, with mean measurements presented in B (values normalized to cell width). ncells, hearts: WT: 5, 3; Ttntv/+: 6, 3. C, Mean passive force measurements for the same cells, assessed as basal force at each step of stretch. D, Proportions of shortening and stretched sarcomeres. E, Magnitude of deformation for shortening and stretched sarcomeres. nsarcomeres: WT: 0% stretch, 103; 5% stretch, 105; 10% stretch, 99; 15% stretch, 106; 20% stretch, 94, from 5 cells, 3 hearts; Ttntv/+: 0% stretch, 99; 5% stretch, 107; 10% stretch, 120; 15% stretch, 117; 20% stretch, 115, from 6 cells, 3 hearts. Data are presented as mean±SEM with sigmoidal curve fitting (B), exponential curve fitting (C), or linear regression (D and E). Statistic methods: (B and C) 2-way repeated measures ANOVA; (D) linear mixed model; (E) Kruskal-Wallis test for within-group comparisons, Mann-Whitney U test for between-group comparisons.…

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  Figure 8.. Increased nonuniformity of sarcomere strain and lowered force production culminate in impaired work performance in TTNtv/+. Changes in TTNtv/+ cardiomyocytes observed experimentally (Figures 6 and 7) were incorporated in the modeled array of 1500 sarcomeres to examine effects of stretch on the uniformity of sarcomere strain, force production, and work performance. Changes included (A) increasing sarcomere length variability by 4%, (B) reducing passive stiffness by 50%, and (C) shortening the A band by 3%. Combining these alterations (D) resulted in a higher proportion of stretched sarcomeres and lowered force and work production following stretch. Similar results were obtained when changes in passive stiffness were excluded (Figure S11).