Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles

Abstract

Residual Force enhancement (RFE) is defined as the increase in steady state force after active stretch compared with force after isometric contraction at the same final length. Recent studies have demonstrated a pivotal role for titin in RFE. In myofibrils stretched beyond overlap of thick and thin filaments, Ca2+ activated myofibrils had a higher titin-based force and a steeper increase with force with stretch than passively stretched myofibrils at the same average sarcomere length. These results suggest higher titin-based stiffness in active myofibrils, perhaps due to N2A titin-actin interactions in active wild type (WT) muscle. Mdm mice carry a small deletion in titin that includes 53 amino acids in Ig83 in the N2A region. It has been proposed that the mdm mutation prevents N2A titin-actin interactions so that active mdm muscles are more compliant than WT muscles. This decrease in active muscle stiffness should be associated with a reduction in RFE. Based on this rationale, we investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from 20-30 day old mdm mice (n=5), harvested at a young age to diminish the effects of degeneration and regeneration, and 30-60 day old WT mice (n=5). Contractile stress and RFE of EDL (mdm n=19 and WT n=20) and SOL fibers (WT n=19 and mdm n=16) was analyzed using JMP and Graph pad (PRISM) software. Statistics included Shapiro-Wilk (normality), Levene's and Bartlett (homogeneity of variances), Welch's one-way ANOVA (unequal variances), Dunnett's T3 (multiple comparisons), and T-tests with Welch's correction (unpaired). To investigate differences in RFE between genotypes, we performed isometric contractions, as well as active and passive stretch from an average sarcomere length of 2.6 - 3.0 µm on each fiber. This stretch amplitude, within the physiological range, resulted in no observable breakage or slipping of fibers during experiments. We predicted that mdm fiber bundles would show reduced RFE compared to WT fibers. Contractile stress when activated at 2.6 µm was significantly greater in WT EDL (Welch's one-way ANOVA; P<0.05) bundle and SOL (Welch's one-way ANOVA; P<0.05) compared to fiber bundles. Our results showed that mdm muscles exhibit significantly lower RFE than WT for both SOL (Welch's one-way ANOVA; P<0.0001) and EDL (Welch's one-way ANOVA; P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. The data support the hypothesis that titin contributes to RFE. We suggest that RFE is reduced in mdm muscles due to impaired Ca2+ dependent titin-actin interactions, resulting from the small deletion in titin that includes 53 amino acids in Ig83 in the N2A region.