FIG4 exhibits an important role in central and peripheral nervous system development and homeostasis. The consequences of FIG4 deficiency on other organ systems are less well delineated. In this study, we defined the consequences of Fig4-null mutation on skeletal muscle. We found that (1) global inactivation of Fig4 in the plt mouse is associated with myofiber atrophy and/or hypotrophy, increased apoptosis and diminished specific force production; (2) loss of Fig4 does not alter the structure, EC coupling apparatus or bidirectional DHPR–RyR1 coupling; and (3) reexpression of Fig4 in neurons significantly reduces all observed muscle abnormalities. The implications of these findings are discussed below.
Perhaps the most striking aspect of this study is the fact that Fig4 mutation did not result in a more deleterious direct effect on muscle development and function. In neurons and glia, loss of FIG4 results in severe structural and functional consequences. Given the previously recognized importance of phosphoinositide regulation in skeletal muscle [26, 27], we predicted that plt mice would exhibit pronounced changes in muscle structure and function. However, effects of Fig4 ablation on muscle were minimal and largely rescued following neuron-specific restoration of FIG4 expression (Fig4
/TgNSE mice). Muscle alterations found to persist in Fig4
/TgNSE mice (for example, 11% reduction in fiber size) could be explained by either a minor role for FIG4 expression in muscle fiber size determination or a small amount of residual neurogenic atrophy. It is of interest to note the dichotomy between the relatively normal appearance of adult plt muscle in situ and the extensive vacuolarization of plt myocytes in vitro. Similar effects of culturing have been observed in other cell types, including fibroblasts  and osteoblasts (unpublished manuscript, Lenk GM and Meisler MH). The explanation for this difference is unclear and requires further experimentation.
There are several potential explanations for the lack of a more severe phenotype in skeletal muscle of Fig4-null mice. First of all, the partial reduction of PI(3,5)P2 likely caused by loss of Fig4 may not be enough to result in significant consequences for muscle development and function. Alternatively, a different PIKfyve protein complex may compensate for the loss of FIG4 to provide sufficient levels of PI(3,5)P2 to maintain myofiber homeostasis. A third possibility is that PI(3,5)P2 may be generated in muscle by a different or complementary pathway. For example, a three-position kinase may generate PI(3,5)P2 from PI(5)P. One current barrier to attempting to distinguish these possibilities is the lack of suitable approaches to measure PI(3,5)P2 in whole tissues such as skeletal muscle. Development of techniques to aid in measuring PI(3,5)P2in situ are required to answer these questions more definitively.
Regardless of the explanation, the fact that there is little, if any, muscle-cell autonomous phenotype in Fig4-null animals indicates that FIG4 mutations are unlikely to result in primary muscle disease. However, significant secondary neurogenically mediated myopathic features, such as those observed in plt mice, including reduced muscle fiber CSA and specific force generation, suggest that skeletal muscle changes may contribute to disease pathogenesis. In other words, the myopathic changes described herein may influence disease severity in patients with Charcot-Marie-Tooth disease type 4J and other disorders caused by FIG4 gene mutation.
The lack of impairment in the structure of the EC coupling apparatus or of bidirectional triad coupling in Fig4-null mice was unexpected. Data from the MTMR14-knockout mice support the hypothesis that increased levels of PI(3,5)P2 impair calcium release from the ryanodine receptor (the core component of the EC apparatus), though the underlying mechanism is not clear. Our data imply that reduced levels of PI(3,5)P2 do not acutely impair voltage-gated triad calcium release. Furthermore, the chronic loss of FIG4 from muscle (with the potential implication of chronically reduced PI(3,5)P2 levels) does not alter the ultrastructural appearance of the triad (that is, the location of the EC coupling machinery). Thus, a requirement for normal levels of PI(3,5)P2 for EC coupling seems unlikely, though the present data do not completely exclude this possibility. In addition to more direct interrogation of EC coupling in FIG4-deficient mice, another potential future direction to address this issue would be to assess the impact on EC coupling of muscle-specific knockout of PIKfyve, the kinase required for PI(3,5)P2 generation. Again, however, this would necessitate confirmation of a specific reduction in PI(3,5)P2 levels in skeletal muscle.
The final significant observation in this study is that reduced Fig4 expression via plt haploinsufficiency does not significantly alter the phenotype of Mtm1-knockout mice. Of note, the MTMR2 gene encodes a protein that is highly homologous to MTM1. In addition, we previously demonstrated that zebrafish mtmr2 functionally compensates (at least in part) for loss of mtm1, suggesting that MTMR2 and MTM1 are functionally quite similar . However, in contrast to our findings in Mtm1- null mice, Bolino and colleagues found that plt haploinsufficiency rescued neuropathy in Mtmr2-knockout animals . The reasons why reduction of FIG4 levels improved the MTMR2-related neuropathology, but not the muscle pathology, seen in Mtm1- null mice are not clear. This distinction may provide another indication of the nonessential role of FIG4 in skeletal muscle or may reflect different quantitative requirements for PI(3,5)P2 in neurons and muscle or specific differences between mammalian MTMR2 and MTM1.