T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase

Abstract

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca(2+) release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca(2+) removal and SR Ca(2+) content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca(2+) release is responsible for the failure of muscle function in myotubular myopathy.

Fig. 1.

Triad disorganization in myotubularin-deficient muscle. (A) Distribution of T-tubules and SR in semithin sections of skeletal muscle from 5-week-old WT and Mtm1 KO mice by immunolabeling of DHPRα, RYR1, and SERCA1. (B) Electron micrographs of WT and KO muscle labeled with the potassium ferrocyanide [K₃Fe(CN)₆] procedure. Electron dense material is located within the lumen of T-tubules (see high magnification in WT and KO micrographs). Upper arrow shows the presence of a L-tubule in mutant muscle. Bottom arrow shows a missing T-tubule/triad. (C) Percentage of L-tubules (2 ± 0.4) in WT and (26 ± 4.8) in KO muscle fibers of mice at 5 weeks of age (P < 0.001). (D) Percentage of tubules per Z-line in myofibers of 5-week-old mice (P < 0.001). (E) Percentage of L-tubules in muscle fibers of mice at 2 weeks of age (3.6 ± 0.3 for WT and 15 ± 1.1 KO, P < 0.001). (F) Percentage of tubules per Z-line in myofibers of 2-week-old mice (P < 0.001). Data were obtained from n = 16 TA muscle fibers of four WT and four KO mice at both 5 and 2 weeks of age.

Fig. 2.

Dysregulation of genes involved in calcium homeostasis in mutant mice. (A) Fold change of DHPRα₁, β₁, and γ₁ subunits (Cacna1s, Cacnb1, and Cacng1), type 1 ryanodine receptor (RyR1), type 1 and 2 SERCA pumps (Atp2a1, Atp2a2), and calsequestrin 1 and 2 (Casq1, Casq2) mRNA levels in KO versus WT TA muscle in 5-week-old mice (n = 3 samples for both WT and KO, P = 0.01 for both Cacnb1 and Casq2, and P = 0.03 for Cacng1; each sample was analyzed as triplicates). (B) Immunoblots show that the protein level of RYR1 and DHPRα₁ are decreased in TA mutant muscle at 5 weeks, whereas DHPRβ₁ level is higher (for DHPRα₁, n = 4, biological samples were analyzed from both WT and KO mice, P = 0.05, and n = 7, TA samples of each genotype were used to quantify the level of the other proteins, P = 0.004 for RYR1 and P < 0.001 for DHPRβ₁).

Fig. 3.

E-C coupling defects in muscle fibers from Mtm1 KO mice. (A) Indo-1 calcium transients elicited by voltage-clamp depolarizations of 5, 10, and 20 ms duration from −80 mV to +10 mV in a WT (left) and KO (right) muscle fiber. (B–D) Graphs show mean values for peak Δ[Ca²⁺], resting [Ca²⁺], and time constant of [Ca²⁺] decay after the end of the pulse for indo-1 transients measured under the same conditions as in panel A (n = 14 and 14, fibers from two WT and two KO mice, respectively).

Fig. 4.

Altered calcium channel function of the DHPR in 5-week-old mutant mice. (A) Ca²⁺ current records measured in response to 0.5-s-long depolarizing pulses to −50, −30, −10, +10, +30, +50, and + 70 mV in a WT (left) and myotubularin-deficient (right) muscle fiber. (B) Mean peak Ca²⁺ current versus voltage relationship of 16 muscle fibers from two WT mice and 20 fibers from two KO mice, under the conditions illustrated in panel A.