Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology

Abstract

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants.

Keywords: Cltc; Snap23; T-tubules; Tmed2; Trip10; alternative splicing; muscle; trafficking.

Figure 1. Modulation of Endogenous AS In Vivo Is Highly Efficient

(A) Quantitation of the percent of inclusion (% incl) by RT-PCR. *p ≤ 0.05 (t test); n = 20–22 (FDB); n = 3 (heart). Results are mean ± SEM. (B) RT-PCR assays on skeletal muscles at postnatal day 2 (PN2) (forelimbs: Trip10; gastrocnemius: rest) and adult stages (triceps: Trip10; gastrocnemius: rest). Panels for each event come from the same gel. (C and D) FDBs were isolated 3 weeks after MO delivery and analyzed by RT-PCR (C) or western blot (D) assays. *p ≤ 0.05 (t test); n = 20–22 (RT-PCRs: Snap23; Trip10; Cltc; Tmed2; and Vps39); n = 8–9 (RT-PCRs: Bin1); n = 3 (western blots). Results are mean ± SEM. n.s., not significant.

Figure 2. AS Modulation Reduces Muscle Force Generation and Calcium Release and Induces Centralized Nuclei and Myofiber Enlargement

(A) Representative calcium tracing following 1-Hz and 150-Hz stimulations (control, black; mix MO, red). Fura 4-F excitation ratio (360/380 nm) and emission (510 nm) were monitored simultaneously with force-frequency characteristics analysis. (B) Representative force tracing following 1-Hz and 150-Hz stimulations (control, black; mix MO, red). Force-frequency characteristics were measured at stimulation frequencies of 1, 10, 20, 40, 80, 100, and 150 Hz every minute. Force was normalized to the CSA. *p ≤ 0.05 (ANOVA); n = 3–4 animals/condition. (C) FDB samples were analyzed by H&E staining and bright field microscopy (10× objective). Percentage of myofibers with centralized nuclei was measured. *p ≤ 0.05; n = 46 (controls) or 28 images (mix MO); three animals/condition. The scale bars represent 50 μm. (D) CSA and minimum Feret’s diameter (min Feret diam). *p ≤ 0.05 (t test); #p ≤ 0.05 (Kruskal Wallis non-parametric test); n = 30–210 fibers/image; 10–14 images/condition; six animals/condition. (E) FDB myofibers were immunostained and imaged by confocal microscopy (10× objective). *p ≤ 0.05 (t test); n = 57–67 fibers/condition; three animals/condition. Results are mean ± SEM or median ± SEM. vol, volume.

Figure 3. T-tubule Organization Is Disrupted in FDB Myofibers after AS Modulation

(A) FDB myofibers were enzymatically isolated, stained with 5 μg/ml FM4-64 fx, fixed, and imaged by confocal microscopy (100× objective). (B and C) Normalized T-tubule power (norm TT power) was defined as the ratio between the first harmonic power and the baseline (B). Distribution histograms are shown (C). (D) Confocal images were used to compute plots along longitudinal lines within the myofibers. We measured the number of picks (T-tubules) per unit length (100 μm). *p ≤0.05 (t test); n = 22–23 fibers/condition; three animals/condition. Results are mean ± SEM. The scale bars represent 20 μm.

Figure 4. Ryr 1 and DHPR Are Mislocalized after AS Redirection

(A) FDB myofibers were immunostained (Ryr1, green; DHPR, magenta). The scale bars represent 20 μm. (B) Colocalization Manders coefficients (M) and frequency plots were obtained on the region of interest (white rectangles in A). The scale bars represent 10 μm. (C) Distribution of Manders coefficients within the myofiber population. *p ≤0.05 (t test); n = 18 (control) or 23 (mix MO) fibers; three animals/condition. Results are mean ± SEM.

Figure 5. Summary

AS redirection of four trafficking events impacts on T-tubule organization, fiber size, Ryr1 and DHPR localization, and colocalization. In consequence, muscle force generation and calcium release are reduced.