Measuring sarcomere dynamics following immunofluorescent labelling of constitutive structural and contractile proteins.

Abstract

Myofibrils are subcellular organelles made of sarcomeres. Characterizing the interaction between constitutive proteins may rationalize mechanisms of muscle contraction. Labelling of titin with immunofluorescent antibodies can reveal segmental elongation of domains while subverting shortcomings including inconsistent intensities and asymmetricity of striations (A-band, I-band) inherent to bright field or phase contrast microscopy methods. Considering antibody labelling has been associated with impairments of sarcomere dynamics, we investigated the effect of labelling α-actinin (Z-disc), myomesin (M-line), and I-band titin epitopes to determine how specific antibodies and solution condition parameters might compromise passive force and sarcomere lengths (SLs). Myofibrils were harvested and purified from psoas of New Zealand White rabbits. Labelled samples were incubated with anti-α-actinin, anti-myomesin, and AlexaFluor488 antibodies. Samples were flushed with relaxing solution, restrained in needle-nanolever systems, and observed under 100x magnification (65 nm/pixel). Myofibrils (labelled, n > 6, 150 sarcomeres; controls n = 7, 141 sarcomeres) were stretched passively from SL ranges of 2.5-2.6 μm (relaxed position; RP) to 3.1-3.25 μm (stretched position; SP). Investigating SPs, forces of 66 ± 22 and 60 ± 23 nN/μm2 were obtained in control and labelled myofibrils. No statistical significance was identified, indicating antibodies did not constrain dynamics in limited passive stretch conditions. Standard deviation of individual SLs decreased from 0.15 to 0.09 μm (39%) and 0.17 to 0.13 μm (24%) in RP1 and SP1. Decrease in SL non-uniformity was pronounced in RPs, indicating antibodies may have stiffened lattice structures to induce subtle friction. SL measurement precision was improved in fluorescent compared to phase-contrast trials by 21%. Antibodies had minimal influence at longer lengths but became increasingly constraining at shorter SLs. These results inform increasingly invasive experiments to identify how titin participates in muscle contraction.