Polycystin-2 Is Required for Starvation- and Rapamycin-Induced Atrophy in Myotubes.

Muscle atrophy involves a massive catabolism of intracellular components leading to a significant reduction in cellular and tissue volume. In this regard, autophagy, an intracellular mechanism that degrades proteins and organelles, has been implicated with muscle breakdown. Recently, it has shown that polycystin-2 (PC2), a membrane protein that belongs to the transient receptor potential (TRP) family, is required for the maintenance of cellular proteostasis, by regulating autophagy in several cell types. The role of PC2 in the control of atrophy and autophagy in skeletal muscle remains unknown. Here, we show that PC2 is required for the induction of atrophy in C2C12 myotubes caused by nutrient deprivation or rapamycin exposure. Consistently, overexpression of PC2 induces atrophy in C2C12 myotubes as indicated by decreasing of the myogenic proteins myogenin and caveolin-3. In addition, we show that inhibition of mTORC1, by starvation or rapamycin is inhibited in cells when PC2 is silenced. Importantly, even if PC2 regulates mTORC1, our results show that the regulation of atrophy by PC2 is independent of autophagy. This study provides novel evidence regarding the role of PC2 in skeletal muscle cell atrophy.

Biological, mechanical and adhesive properties of universal adhesives containing zinc and copper nanoparticles.

OBJECTIVES: To evaluate the effect of addition of zinc oxide and copper nanoparticles (ZnO/CuNp) into universal adhesives, on antimicrobial activity (AMA), cytotoxicity (CTX), water sorption (WS) and solubility (SO), microhardness (MH) and in vitro degree of conversion (DC), as well as resin-dentin microtensile bond strength (µTBS), nanoleakage (NL) and in situ DC. METHODS: ZnO/CuNp (0% [control]; 5/0.1 and 5/0.2 wt%) were added in Prime&Bond Active (PBA) and Ambar Universal (AMB). The AMA was evaluated against Streptococcus mutans. For CTX, Saos-2 cell-line was used. For WS and SO, specimens were tested for 28d. For MH, specimens were tested after 24 h and 28d and for in vitro DC, specimens were evaluated after 24 h. After, the adhesives were applied to flat dentine surfaces, composite resin build-ups, specimens were sectioned to obtain resin-dentine sticks. It was evaluated in µTBS, NL and in situ DC after 24 h of water storage. ANOVA and Tukey's test were applied (α = 0.05). RESULTS: The addition of 5/0.2 ZnO/CuNp increase AMA and WS, but decrease the SO when compared to control (p < 0.05). The CTX and µTBS were maintaining with adhesive-containing ZnO/CuNp (p > 0.05). MH, in vitro DC and in situ DC was significant increase (AMB) or maintaining (PBA) with ZnO/CuNp addition. However, significantly lower NL was observed for ZnO/CuNp groups (p < 0.05). CONCLUSIONS: The addition of ZnO/CuNp in the tested concentrations in universal adhesive systems may be an alternative to provide antimicrobial activity and improves the integrity of the hybrid layer, without jeopardizing biological, adhesives and mechanical properties. SIGNIFICANCE: This is the first study that demonstrates that the addition of zinc oxide and copper nanoparticles in concentrations up to 5/0.2 wt% in two universal adhesive systems is a feasible approach and may be an alternative to adhesive interfaces with antimicrobial properties and less defects in the resin-dentin interface.

Early molecular response and microanatomical changes in the masseter muscle and mandibular head after botulinum toxin intervention in adult mice.

BACKGROUND: Masseter muscle paralysis induced by botulinum toxin type A (BoNTA) evokes subchondral bone loss in mandibular heads of adult rats and growing mice after 4 weeks. However, the primary cellular and molecular events leading to altered bone remodeling remain unexplored. Thus, the aim of the current work has been to assess the molecular response that precedes the early microanatomical changes in the masseter muscle and subchondral bone of the mandibular head in adult mice after BoNTA intervention. METHODS: A pre-clinical in vivo study was performed by a single intramuscular injection of 0.2 U BoNTA in the right masseter (experimental) of adult BALB/c mice. The contralateral masseter was injected with vehicle (control). Changes in mRNA levels of molecular markers of bone loss or muscle atrophy/regeneration were addressed by qPCR at day 2 or 7, respectively. mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) was assessed in mandibular heads, whilst mRNA levels of Atrogin-1/MAFbx, MuRF-1 and Myogenin were addressed in masseter muscles. In order to identify the early microanatomical changes at day 14, fiber diameters in transversal sections of masseter muscles were quantified, and histomorphometric analysis was used to determine the bone per tissue area and the trabecular thickness of subchondral bone of the mandibular heads. RESULTS: An increase of up to 4-fold in RANKL mRNA levels were detected in mandibular heads of the BoNTA-injected sides as early as 2 days after intervention. Moreover, a 4-6 fold increase in Atrogin-1/MAFbx and MuRF-1 and an up to 25 fold increase in Myogenin mRNA level were detected in masseter muscles 7 days after BoNTA injections. Masseter muscle mass, as well as individual muscle fiber diameter, were significantly reduced in BoNTA-injected side after 14 days post-intervention. At the same time, in the mandibular heads from the treated side, the subchondral bone loss was evinced by a significant reduction in bone per tissue area (-40%) and trabecular thickness (-55%). CONCLUSIONS: Our results show that masseter muscle paralysis induced by BoNTA leads to significant microanatomical changes by day 14, preceded by molecular changes as early as 2 days in bone, and 7 days in muscle. Therefore, masseter muscle atrophy and subchondral bone loss detected at 14 days are preceded by molecular responses that occur during the first week after BoNTA intervention.