Gaining new understanding of sarcomere length non-uniformities in skeletal muscles

Figures

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  FIGURE 1. (A) Sarcomere length measurement: An isolated single myofibril was readied for mechanical assessment (A), along with the corresponding dark-light intensity patterns caused by sarcomere structures. The dark A-bands and Z-lines show high intensity, while the light I-bands show a low intensity (B). The loss of intensity in the middle of the A-band indicates the H-zone, that is the region in the centre of the A-band that does not contain actin filaments. The length of a sarcomere was measured by the distance of adjacent A-band centroids, the location of which is indicated by the dashed vertical lines, see SL1 and SL2. Adapted from Schmidt et al. (2021); with permission from Elsevier. (B) Experimental Protocol: Sarcomere lengths were measured at seven different instances during the experimental protocol that involved three activation-deactivation cycles. The total myofibril length remained unchanged throughout these cycles. Yellow dots indicate the relaxed states with sarcomere length measurements made prior to activation (P0) and following the three activation-deactivation cycles (P1, P2 and P3, respectively); Red dots indicate the active states with measurements made during the…

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  FIGURE 2. Activation (going from the relaxed to the activated state) produced an increase in sarcomere lengths non-uniformities at each average sarcomere length that was tested but repeat activation/deactivation did not change sarcomere length non-uniformity in the passive conditions (P0 to P3) or the active conditions (A1 to A3). (A) Sarcomere length non-uniformity expressed as mean standard deviations expressed in µm. (B) Sarcomere length non-uniformity expressed as mean coefficients of variation expressed in percent. The absolute sarcomere length non-uniformities were greatest for the longest average sarcomere lengths (3.6 µm) tested (A), but the coefficients of variation were greatest for the shortest (2.7 µm) test group of activated myofibrils (B).

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  FIGURE 3. Sarcomere lengths of the original passive state (P0 = before the first activation) vs. sarcomere lengths in the passive/relaxed state after the first, second and third activation/deactivation cycles (P1–panel (A); P2–panel (B); and P3–panel (C), respectively). Red squares are sarcomeres from the short group (nominal average sarcomere lengths of 2.7 µm). Blue squares are from the middle group (nominal average sarcomere length of 3.2 µm). Green squares are from the long group (nominal average sarcomere length of 3.6 µm). Yellow squares are sarcomeres whose length changes are greater than the reliability of measurement off 0.2 µm. The solid line represents the best fitting linear regression that is forced through the origin (0/0) of the coordinate system. The dotted lines are offset from the best fitting regression line by ±0.2 µm, which represents the day-to-day reliability of sarcomere length measurements. In other words, sarcomeres within these lines are considered to be of the same length. p < 0.001, there is a very statistically significant relationship between the variables.

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  FIGURE 4. Sarcomere lengths for the first active state (A1 = first activation) vs. sarcomere lengths in the active state following the second and third activation/deactivation cycles (A2 – panel (A); A3 – panel (B), respectively). Red circles are sarcomeres from the short group (nominal average sarcomere length of 2.7 µm). Blue circles are from the middle group (nominal average sarcomere length of 3.2 µm). Green circles are from the long group (nominal average sarcomere length of 3.6 µm). Yellow circles are sarcomeres whose length changes were greater than the day-to-day reliability of ±0.2 µm. The solid line represents the best fitting linear regression that was forced through the origin (0/0) of the coordinate system. The dotted lines are offset from the best fitting regression line by ±0.2 µm, which represents the day-to-day reliability of sarcomere length measurements. Sarcomeres within these dotted lines are considered to be of the same length. p < 0.001, there is a very statistically significant relationship between the variables.

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  FIGURE 5. Sarcomere lengths of the first active state (A1) vs. sarcomere lengths of the initial passive state (P0–panel (A). Sarcomere lengths of the second active state (A2) vs. sarcomere lengths in the passive state after the first activation (P1–panel (B). Sarcomere lengths of the third active state (A3) vs. sarcomere lengths in the passive state after the second activation (P2–panel (C)). Red symbols are sarcomeres from the short group (nominal average sarcomere lengths of 2.7 µm). Blue symbols are from the middle group (nominal average sarcomere length of 3.2 µm). Green symbols are from the long group (nominal average sarcomere length of 3.6 µm). The solid lines of red, blue, green represent the best fitting linear regression for the short group, middle group, and long group myofibrils respectively.

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  FIGURE 6. (A) Sarcomere length changes in the second activation cycle (going from the initial passive state “P0” to the second active state “A2”) as a function of the sarcomere length changes occurring during the first activation cycle (going from the initial passive state “P0” to the first active state “A1”), and (B) the corresponding sarcomere length changes going from the initial passive state “P0” to the third active state “A3” as a function of the length change in the first activation cycle (going from “P0” to “A1”). Data points in the first and third quadrant indicate that sarcomeres were shortening/elongating in both activation cycles, while data points in the second and fourth quadrant indicate that sarcomeres were elongating/shortening or shortening/elongating in the first and subsequent activation cycle. The proportion of sarcomeres falling into the four quadrants are indicated in the red, blue, and green numbers for the short (2.7 µm), middle (3.2 µm) and long (3.6 µm) average sarcomere length tests. The vertical and horizontal lines through the zero values indicate zero length change of a sarcomere in the activation cycle going from the passive to the active state. The…