The distinctive mechanical and structural signatures of residual force enhancement in myofibers

Abstract

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

Fig. 1.. X-ray diffraction of TC fiber bundles before and 50% titin cleavage during Iso_S, Iso_L, and Iso_R conditions.

(A) Schematic of skeletal half-sarcomere with relevant structural periodicities, I-band titin segments, and TEV_P cleavage site indicated. (B-C) Tension traces of fibers during mechanical experiments before (B) and after (C) 50% titin cleavage. Active tension is total tension during contractions, minus the passive tension at those lengths. (D) Passive stiffness after titin cleavage, normalized to its paired pre-stiffness value at Iso_S. (E) Tension before (blue) and after (red) titin cleavage for passive, active, and total conditions. (F) transmission electron micrographs of TC sarcomeres after full mechanical protocols with sham (−TEV_P) or treatment (+TEV_P) conditions. (G) RFE in fibers before and after titin treatment. (H) Representative X-ray diffraction pattern of skeletal psoas fibers, with labeled reflections indicating relevant periodic structures that are referenced in (A). Connecting letters: different letters are significantly different (post hoc analysis P < 0.05). Data throughout reported as mean ± s.e.m. Full statistical details in Table S1. letters are significantly different (post hoc analysis P < 0.05). Data throughout reported as mean ± s.e.m. Full statistical details in Table S1.

Fig. 2.. Sarcomeric structural parameters of TC fibers before and after 50% titin cleavage.

D_1,0 (A), I_1,1/I_1,0 (B), σ_A (C), σ_D (D) S_M3 (E), I_M3 (F), S_gActin (G), S_T3 (H), and S_M6 (I) were recorded before (blue) and after (red) 50% titin cleavage, at three conditions: Iso_S, Iso_L, and Iso_R. Connecting letters: different letters are significantly different (Tukey HSD P < 0.05). Full statistical details in Table S2 and Table S3. Intra-sample pre-post differences in Fig. S3.

Fig. 3.. Titinopathic mdm fibers produce no distinctive RFE condition.

D_1,0 (A), S_M6 (B), I_1,1/I_1,0 (C), σ_A (D), S_M3 (E), and S_A6 (F) were recorded for WT (blue) and mdm (red) EDL fiber bundles at three conditions: Iso_S, Iso_L, and Iso_R. Connecting letters: different letters are significantly different (Tukey HSD P < 0.05). Full statistical details in Table S4.

Fig. 4.. Mechanism of RFE.

Configuration of sarcomeric proteins during (A) Iso_S, (B) Iso_L, and (C) Iso_R that can account for their distinctive mechanical and structural signatures. In passive muscle, low-level titin-thin filament interactions occur in such a way that passive stretch is enough to detach-reattach and/or drag titin along the thin filament so that titin-based free length and extension are still similar to if they were not attached at all. During contraction, the titin-thin filament interaction becomes stronger, so that during an eccentric contraction, titin extension occurs above that in passive, producing elevated titin-based force and explaining the mechanical and structural signatures in Iso_R. Increased titin-based force contributes to RFE, which also leads to smaller lattice spacing and increased thick filament stiffness, improving force production and force transmission, respectively.