Conditional Knockout of Pik3c3 Causes a Murine Muscular Dystrophy

Abstract

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models

“…We have previously shown that muscle-specific knockout of Pik3c3 with Ckmm-Cre results in a significant decrease in total skeletal muscle PI3P levels (18). In our Acta1-Cre KOs, there was again a trend of reduced total PI3P, though with our current sample size it did not reach statistical significance (Supplemental Figure 4D).…”

“…Given that loss of Pik3c2b by itself in mice has no discernible phenotype mtm1 does not significantly affect autophagy, but that secondary impairment in autophagic flux in mtm1 morphants promotes a dramatic worsening of the phenotype (22), and also shown that knockout in muscle of Pik3c3 significantly impairs autophagy (18). Thus a similar phenomenon may be at play in our Pik3c3 Mtm1 dKO mice.…”

“…Comparisons were done using ANOVA followed by Dunnett's multicomparison test. We previously reported that inhibition of autophagy significantly worsened the phenotype in an MTM zebrafish model (22), and have demonstrated that Pik3c3 knockout is associated with impaired autophagy (18). We thus were interested to examine autophagy in Pik3c3 fl/+ Mtm1 KO animals.…”

“…In muscle, PI3P is synthesized by both PIK3C3 and PIK3C2B. We previously created muscle-specific Pik3c3 KO mice using Ckmm-Cre, but these animals die of complications related to severe cardiomyopathy (18). In order to test the effect of Pik3c3 reduction on Mtm1 KO animals, we re-derived the Pik3c3 animals with Cre driven by the Acta1 promoter to confine targeting to skeletal muscle (Supplemental Figure 4A).…”

“…We therefore examined the kinases responsible for generating PI3P (i.e., the class II and class III PI3Ks). We have previously shown that only PIK3C2B (a class II kinase) and PIK3C3 (the class III kinase) are appreciably expressed in skeletal muscle (18), the primary tissue affected in MTM, and have generated and characterized Pik3c3 muscle-specific KO mice. These animals have reduced levels of PI3P in skeletal muscle and manifest a severe cardiomyopathy, an autophagic vacuolar myopathy, and early lethality (18).…”