VCP associated disease results in primarily proximal vacuolar inclusion body myopathy, Paget’s disease of the bone, and FTD [6
]. To date, several disease mutations have been reported, all of which are missense mutations causing an amino acid change in the N-terminal part of the polypeptide [22
]. The high evolutionary conservation suggests an important role for the VCP protein in the normal cellular functions. This is further supported by the finding that homozygous knock-out mice lacking both VCP alleles die in early embryogenesis [23
]. On the other hand, the transgenic mice over-expressing the R155H mutation show muscle weakness and muscle tissue pathology similar to human IBMPFD patients [24
]. The pathological mechanisms resulting in the clinical and cellular phenotypes in the muscle tissues of the human and mouse are still to be resolved. To begin to resolve these mechanisms, we have analyzed human primary myoblast cell lines. By analyzing patients’ cells that express the mutant VCP at an endogenous level, one can preclude the possibility of over-expression artifacts, and therefore these cells can be considered a better model to study cellular pathogenesis of the disease than over-expressing cell models.
Immunocytochemical results showed that the mutant protein is expressed at the similar level to the wild-type protein, and is targeted to the cytoplasm but for an unknown reason it cannot fulfill its proper cellular function at its final destination. This hypothesis is further supported by earlier studies showing normal hexamer formation and normal to elevated ATPase activities for the mutant VCP protein [16
]. Therefore, it is possible that synthesis, processing and targeting of the mutant polypeptides are intact but when hexamers with at least one mutant molecule are formed, their function is severely affected resulting in defective cellular processes. The generation of a knock-out mouse model having only one functional VCP gene also favors this hypothesis, since heterozygous mice having one wild-type allele and one deleted allele are indistinguishable from their wild-type littermates [23
Vacuolization has previously been reported in many cell types (COS, HeLa and PC12) over-expressing the VCP mutant K524A, which affects the ATPase activity [25
]. Here we report the findings of large intracellular vacuoles in mutant myoblast cultures. Ubiquitin or VCP did not accumulate in the vacuoles but other, still unidentified molecules fill the lumen of these organelles. Similar cellular pathology has been reported in Danon disease, another genetic disease characterized by myopathy with vacuolar accumulation seen in 10% of the skeletal muscle fibers [26
], heart disease, and mental retardation [27
]. This disease is caused by mutations in the Lamp2 gene that encodes a lysosomal membrane protein [28
]. Danon disease is considered an autophagic vacuolar myopathy showing intracytoplasmic vacuoles, which are believed to be autolysosomes [29
]. These similarities in clinical and cellular phenotypes urged us to analyze patients’ myoblasts in regard to lysosomal membrane proteins. The Western blotting analysis showed increased molecular weights for Lamp1 and Lamp2 in mutant myoblast cells, which was due to defective N-glycosylation processes of these two proteins. Based on these findings, we hypothesize that the N-glycosylation of Lamp-proteins is disturbed in VCP disease patients’ myoblasts, which may be involved in the defective degradation of vacuolar contents.
Myotube formation is a multi-step process that is regulated by two types of fusion events [30
]. First, myoblasts fuse together to form myotubes containing few nuclei. Subsequently, additional fusion between myoblasts and nascent myotubes results in the formation of large myotubes containing multiple nuclei. These myoblast fusion processes can be studied by analyzing the expression of proteins that are specific for myotubes. Our Western blotting analyses using antibodies raised against myotube specific proteins troponin C and myogenin suggest that the VCP disease mutations result in defective cell fusion processes in myoblast cultures. The observed defective cell fusion may be caused by affected expressions of cell surface proteins. We tested this hypothesis by analyzing the expression of the transmembrane cell adhesion molecule M-cadherin, of which intact expression is required for normal myoblast fusion. M-cadherin is predominantly expressed in developing skeletal muscle, and in mature skeletal muscle it is detectable in satellite cells and on the sarcolemma of myofibers underlying satellite cells [31
]. The observed significant down-regulation of M-cadherin in patients’ myoblasts suggests that it may play an important role in the defective myotube formation in the mutant myoblasts. However, we cannot exclude the possibility of the involvement of other cell adhesion molecules in this multi-step process of myotube formation. Similar findings are seen in Oculopharyngeal muscular dystrophy (OPMD), which is an adult-onset autosomal dominant disease associated with drooping of eyelids and swallowing problems around the age of 50 years. The disease is associated with an expansion in the GCG trinucleotide repeat of the nuclear poly(A)-binding protein (PABPN1) gene [34
]. The hallmark of OPMD is the presence of intranuclear inclusions in the skeletal muscles of patients, and ectopic expression of the mutant PABPN1 produced inclusions in a muscle cell culture model and reduced expression of several muscle-specific proteins including α-actin, troponin C, and myogenic transcription factors, myogenin and MyoD [35
Lastly, we analyzed autophagy and the fate of cultured myoblasts. Autophagy is a process that degrades long-lived proteins and cytoplasmic components within vesicles which deliver the contents to the lysosome/vacuole for degradation. Upon activation of autophagy, the 18 kDa cytosolic LC3 (LC3-I) undergoes proteolytic cleavage followed by a lipid modification and is converted to the 16 kDa membrane-bound form (LC3-II), which is specifically localized to the autophagosomal membranes [36
]. The conversion from LC3-I to LC3-II can be used as a sensitive marker for distinguishing autophagy in mammalian cells. The increased LC3-II form in mutant myoblasts cultured in starvation condition suggests that mutant cells are prone to the increased level of autophagy, and muscle cell defects can be worsened by insufficient nutrition. Increased autophagy has also been reported in another myopathy, hereditary inclusion body myopathy (hIBM), which is caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). The analyses of patients’ tissues revealed that the observed cellular organelles represent autophagic vacuoles [37
]. Based on these similarities between IBMPFD and hIBM, it is likely that these two adult-onset myopathies share similar pathological pathways, and the vacuolization plays an important role in the development of muscle weakness. The defective processing of Lampproteins in patients’ myoblasts may be connected to the increased vacuolization. This hypothesis is supported by the observation that the mice that are double deficient in Lamp1 and Lamp2 show a much higher frequency of cytoplasmic vacuoles, which are often identified as autophagic vacuoles [39
]. It is possible that although the initial maturation of autophagosomes including the fusion with endosomes is functional, the final maturation steps of late autophagic vacuoles may be retarded.
The Caspase-3 assay and TUNEL staining demonstrated increased apoptosis in patients’ myoblasts. This may indicate that the accumulation of storage material in enlarged vacuoles and defective autophagy in patients’ myoblasts is harmful to the normal functioning of differentiated muscle cells. This, in turn, may result in increased apoptosis of patients’ myoblasts. Myotubes are terminally differentiated cells that have lost their ability to proliferate, and therefore the dysfunctions of their cellular processes make them prone to cell death. Similar hypothesis has been proposed for a group of genetic neurodegenerative diseases, e.g. Neuronal Ceroid Lipofuscinoses (NCL) (for review see [40
]), which are characterized by accumulation of storage material in patients’ cells. Despite the accumulation of inclusions in many cell types, only neurons are affected resulting in increased apoptosis of neurons in patients’ central nervous system, whereas other, still dividing cells seem to be unaffected. Thus, both IBMPFD and NCL are characterized by accumulation of storage material in terminally differentiated cells types, which results in death of patients’ cells and manifestation of clinical symptoms later in the progression of the disease.
In conclusion, we have shown that the VCP mutations result in vacuolization of patients’ myoblasts. These vacuoles are, for unknown reason, unable to degrade the vacuolar contents. We also observed that the vacuolization increases with the age of the mutant cells. This possibly results in disturbed cellular processes in patients’ muscle cells including defective myotube formation, increased apoptosis, and increased autophagy (). These defects may partially explain the observed myopathy in patients with VCP associated disease. Therefore, patients’ myoblasts demonstrate a useful cell model that can be utilized in our future studies to clarify the detailed molecular pathways resulting in myopathy and also for the development of novel treatments to alleviate these pathological manifestations in IBMPFD patients.
Potential pathogenesis of the IBMPFD muscle