Murine Fig4 is dispensable for muscle development but required for muscle function

Abstract

Background: Phosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation-contraction coupling. FIG4 functions as a PIP phosphatase that participates in both the synthesis and breakdown of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Mutation of FIG4 results in a severe neurodegenerative disorder in mice and a progressive peripheral polyneuropathy in humans. The effect of FIG4 mutation on skeletal muscle has yet to be examined.

Methods: Herein we characterize the impact of FIG4 on skeletal muscle development and function using the spontaneously occurring mouse mutant pale tremor (plt), a mouse line with a loss of function mutation in Fig4.

Results: In plt mice, we characterized abnormalities in skeletal muscle, including reduced muscle size and specific force generation. We also uncovered ultrastructural abnormalities and increased programmed cell death. Conversely, we detected no structural or functional abnormalities to suggest impairment of excitation-contraction coupling, a process previously shown to be influenced by PI(3,5)P2 levels. Conditional rescue of Fig4 mutation in neurons prevented overt muscle weakness and the development of obvious muscle abnormalities, suggesting that the changes observed in the plt mice were primarily related to denervation of skeletal muscle. On the basis of the ability of reduced FIG4 levels to rescue aspects of Mtmr2-dependent neuropathy, we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Male mice with a compound Fig4+/-/Mtm1-/Y genotype displayed no improvements in muscle histology, muscle size or overall survival, indicating that FIG4 reduction does not ameliorate the Mtm1-knockout phenotype.

Conclusions: Overall, these data indicate that loss of Fig4 impairs skeletal muscle function but does not significantly affect its structural development.

Figure 1

FIG4 is expressed in skeletal muscle. Western blot analysis was performed to establish FIG4 expression using anti-FIG4 antibody. (A) Mouse multitissue Western blot reveals expression of FIG4 in a variety of tissues (Br = brain, Ht = heart, SmI = small intestine, Kid = kidney, Liv = liver, Lg = lung, SkM = skeletal muscle, St = stomach, Spl = spleen, Ov = ovary, Tes = testis). (B) FIG4 is expressed at multiple mouse ages in brain and skeletal muscle. Ages of mice tested were 1, 3, 6, 9 and 20 months. Left lanes are from brain, and right lanes are from quadriceps. The top blot was probed with anti-FIG4, and the bottom blot (loading control) was probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (C) Western blot of protein extracts from C2C12 cells at various stages of differentiation. Differentiation was induced by serum withdrawal. Cells were differentiated until long myotubes were obviously present (day 8 = D8). (D) Analysis of wild-type littermate and pale tremor (plt) mouse skeletal muscle reveals that FIG4 is absent from plt muscle.

Figure 2

Histopathological analysis of pale tremor (plt) skeletal muscle. Hematoxylin and eosin staining (A) and (B) and succinate dehydrogenase staining (C) and (D) of cryosections from quadriceps of control (wild-type (WT)/littermate (LM)) and plt animals. Scale bars = 50 μm. (E) Quantitation of myofiber size from quadriceps muscle of 4-week-old plt and WT animals. There were significant reductions in myofiber size: 2,649 μm² ±70 for WT vs 1,185 μm² ± 32 for plt (n = 400 per condition, P < 0.0001).

Figure 3

Ultrastructural analysis of pale tremor (plt) skeletal muscle. Representative photomicrographs from transmission electron microscopic analysis of wild-type/littermate (WT) quadriceps (A) and plt quadriceps (B) and (C). Muscle ultrastructure was generally normal in plt muscle (B) with evidence of normal triads (arrow). There were infrequently observed areas of abnormalities that particularly included swollen and/or dilated mitochondria (M in (C)). Scale bars = 500 nm.

Figure 4

Increased apoptosis in plt skeletal muscle and skeletal myocytes. (A) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed on sections from wild-type (WT) and plt skeletal muscle. Arrows point to TUNEL-positive nuclei. There was a qualitative increase in TUNEL-positive fibers in plt muscle (experiment repeated with sections from three different mice per condition). (B) TUNEL staining was performed on myocytes. There was a significant increase in TUNEL-positive cells in plt myocytes vs WT myocytes. Data reflect fraction of 4′,6-diamidino-2-phenylindole-positive nuclei that were also TUNEL-positive. Numbers were 0.28 ± 0.06 for plt and 0.17 ± 0.05 for WT (P = 0.006). One hundred cells per experiment were counted, and each experiment was repeated five times (once for each culture derived with cells isolated from five different mice per condition).

Figure 5

Diminished force production in pale tremor (plt) skeletal muscle. Specific force (normalized to muscle and body weight) was measured for wild-type/littermate (WT) and plt (mutant) skeletal muscle (left panel = extensor digitorum longus, or EDL) (right panel = soleus). Normalized force was significantly reduced in plt muscles (n = 7 animals tested per condition). *P = 0.043 for EDL and P < 0.001 for soleus.

Figure 6

L-currents and voltage-gated Ca²⁺ release are unaltered in pale tremor ( plt ) myotubes. (A) Representative whole-cell L-type Ca²⁺ currents (I_Ca) and intracellular Ca²⁺ transients (ΔF/F) obtained following 200-ms depolarizations to the indicated membrane potentials in myotubes derived from either wild-type (WT) mice (upper panel) or plt mice (lower panel). (B) and (C) Average (±SEM) voltage dependence of peak L-type Ca²⁺ current density (B) and intracellular Ca²⁺ transients (C) for myotubes derived from either WT mice (filled circles) or plt mice (open circles).

Figure 7

Transgenic (Tg) neuronal rescue of FIG4 expression largely restores plt myofiber size. (A) Hematoxylin and eosin–stained sections of gastrocnemius muscle from wild-type (WT) and Fig4^−/−/NSE-FIG4 (that is, plt with transgenic expression of Fig4 driven by the neuron-specific enolase (NSE) promoter). The plt muscle is essentially indistinguishable from WT, with the exception of mild reduction in fiber size. (B) Quantification of myofiber size from quadriceps muscles of 8-month-old Fig4^−/−/NSE-FIG4 and WT animals. There was a small but significant reduction in total fiber area in transgenically rescued plt muscle: 2,799 μm² ± 165 for WT vs 2,322 μm² ± 121 for plt (n = 60, P = 0.02). (C) Transmission electron photomicrographs from tibialis anterior muscle. Normal triads and mitochondria were present in both WT and plt/Tg-NSE-Fig4 animals.

Figure 8

Fig4 haploinsufficiency does not improve the Mtm1-knockout phenotype. (A) Hematoxylin and eosin stain of quadriceps muscle from wild-type (WT; plt^+/−) (top), Mtm1-knockout (Mtm1-KO) (SM, middle) and plt^+/−/Mtm1-KO (DM, bottom) animals. There was no obvious difference in the appearance of the muscle or in the number of abnormal fibers (arrows). (B) Distribution of fiber area. Fiber area was measured from WT; plt^+/−), single-mutant (SM; Mtm1-KO) and double-mutant (DM; plt^+/−/Mtm1-KO) skeletal muscles. Thirty fibers from each animal were measured (n = 4 for SM and DM, n =2 for WT). (C) Terminal body weight (measured in grams). There was significant reduction in the Mtm1-KO body weight compared to WT. There was no improvement in body weight in plt^+/−/Mtm1-KO animals. Average values were as follows: 17.8 ± 0.6 g for WT (n = 8), 11.4 ± 0.7 g for Mtm1-KO (n = 9, P < 0.0001 compared to WT) and 10.5 ± 0.9 g for plt^+/−/Mtm1-KO (n = 5, P = 0.48 compared to Mtm1-KO). (D) Haploinsufficiency of Fig4 did not provide any survival benefit. Average survival was 32 ± 2 days for Mtm1-KO (n = 9) and 31 ± 3 days for plt^+/−/Mtm1-KO (n = 5).