Mannose-methyl-ß-cyclodextrin suppresses tumor growth by targeting both colon cancer cells and tumor-associated macrophages.

Methylated ß-cyclodextrin (MßCD) can extract cholesterol from lipid rafts and induce apoptosis in cancer cells by inhibiting activation of the PI3K-Akt-Bad pathway. In this study, we modified MßCD with mannose (Man-MßCD) and assessed its in vitro and in vivo potential for targeting colon cancer cells expressing the mannose receptor (MR) and tumor-associated macrophages (TAM). Man-MßCD showed a significantly greater level of cellular association with colon-26 cells and M2 macrophages, and much more prominent anticancer activity than that of MßCD against MR-positive colon-26 cells. These results revealed that autophagy was the main mechanism of cell death associated with Man-MßCD. Furthermore, compared with MßCD, Man-MßCD significantly reduced tumor development following intravenous delivery to tumor-bearing mice, with no apparent side effects. Thus, Man-MßCD has the potential to be a novel anticancer drug.

Antigen stabilizing hydrogels based on cyclodextrins and polyethylene glycol act as type-2 adjuvants with suppressed local irritation.

Recent viral pandemics have increased global demand for vaccines. However, the supply of effective and safe vaccine not only to developed countries but also developing countries with inadequate storage equipment is still challenging due to the lack of robust systems which improve the efficacy and the stability of vaccines with few side effects. In our previous study, polypseudorotaxane (PPRX) hydrogels based on cyclodextrin (CyD) and polyethylene glycol (PEG) significantly improved the stability of antibody preparations and showed no serious adverse effects after subcutaneous injection, suggesting the possibility as safe vaccine formulations to stabilize an antigen protein. Moreover, recent studies have reported that one of the CyD derivatives, hydroxypropyl-ß-CyD (HP-ß-CyD), acts as an adjuvant to enhance protective type-2 immune responses. However, it is still unknown that CyD PPRX hydrogels enhance not only the stability of an antigen protein but also its immunogenicity with tolerable side effects. Here, we demonstrate that α- and γ-CyD PPRX hydrogels containing an antigen protein significantly induce antigen-specific type-2 immune responses. Moreover, α- and γ-CyD PPRX hydrogels showed negligible local irritation at the injection site, although subcutaneous injection of α-CyD alone induced skin lesion. Finally, shaking stability of the antigen protein at room temperature was significantly improved by being included in α- and γ-CyD PPRX hydrogels. These results propose the possibility of α- and γ-CyD PPRX hydrogels as novel vaccine formulations which improve both the immunogenicity and stability of an antigen protein with suppressed local irritation.

Improvement of Pharmaceutical Properties of Zerumbone, a Multifunctional Compound, Using Cyclodextrin Derivatives.

Zerumbone is a multifunctional compound which shows various biological activities, such as antitumor activity, anti-inflammatory activity, antiulcer activity, etc. However, to use Zerumbone as functional foods or medicines, its pharmaceutical properties such as solubility should be improved. In the present study, we prepared its inclusion complexes with various cyclodextrin (CyD) derivatives, and evaluated their solubility, release profile of the drug and cytotoxic activity. Among 11 CyDs, sulfobutylether (SBE)-ß-CyD showed the highest solubilizing effect for Zerumbone. Phase solubility diagrams of SBE-ß-CyD/Zerumbone in 10% methanol solution showed AL type, and the stability constant was 756 M-1. SBE-ß-CyD also formed the solid complex with Zerumbone by kneading for 90 min. Importantly, the dissolution rate of Zerumbone was improved by complexation with SBE-ß- and hydroxypropyl (HP)-ß-CyDs, and its supersaturation was maintained for several hours. The solubilizing effects by SBE-ß-CyD was greater than that of HP-ß-CyD. Moreover, SBE-ß-CyD/Zerumbone complex also retained the cytotoxic activity of Zerumbone. These results suggest that CyDs, especially SBE-ß-CyD, were useful to improve the solubility of Zerumbone.

Lactose-appended ß-cyclodextrin as an effective nanocarrier for brain delivery.

The blood-brain barrier (BBB) prevents the permeability of drugs into the brain, and as such limits the management of various brain diseases. To overcome this barrier, drug-encapsulating nanoparticles or vesicles, drug conjugates, and other types of drug delivery systems (DDSs) have been developed. However, the brain-targeting ability of nanoparticles or vesicles is still insufficient. Recently, among the various brain-targeting ligands previously studied for facilitating transcellular BBB transport, several sugar-appended nanocarriers for brain delivery were identified. Meanwhile, cyclodextrins (CyDs) have been used as nanocarriers for drug delivery since they can encapsulate hydrophobic compounds with high biocompatibility. Therefore, in this study, we created various sugar-appended ß-cyclodextrins (ß-CyDs) to discover novel brain-targeting ligands. As a result, of the six sugar-appended CyDs, lactose-appended ß-CyD (Lac-ß-CyD) showed greater cellular uptake in hCMEC/D3 cells, human brain microvascular endothelial cells, than other sugar-appended ß-CyDs did. In addition, the permeability of Lac-ß-CyD within the in vitro human BBB model was greater than that of other sugar-appended ß-CyDs. Moreover, Lac-ß-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-ß-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-ß-CyD has the potential to be a novel carrier for drugs across the BBB.

MBNL1-Associated Mitochondrial Dysfunction and Apoptosis in C2C12 Myotubes and Mouse Skeletal Muscle.

We explored the interrelationship between a tissue-specific alternative splicing factor muscleblind-like 1 (MBNL1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), B-cell lymphoma 2 (Bcl-2) or Bcl-2-associated X protein (Bax) in C2C12 myotubes and mouse skeletal muscle to investigate a possible physiological role of MBNL1 in mitochondrial-associated apoptosis of skeletal muscle. Expression level of PGC-1α and mitochondrial membrane potential evaluated by the fluorescence ratio of JC-1 aggregate to monomer in C2C12 myotubes were suppressed by knockdown of MBNL1. Conversely, the ratio of Bax to Bcl-2 as well as the apoptotic index in C2C12 myotubes was increased by MBNL1 knockdown. In plantaris muscle, on the other hand, not only the minimum muscle fiber diameter but also the expression level of MBNL1 and PGC-1α in of 100-week-old mice were significantly lower than that of 10-week-old mice. Furthermore, the ratio of Bax to Bcl-2 in mouse plantaris muscle was increased by aging. These results suggest that MBNL1 may play a key role in aging-associated muscle atrophy accompanied with mitochondrial dysfunction and apoptosis via mediating PGC-1α expression in skeletal muscle.

The Protective Effect of Brazilian Propolis against Glycation Stress in Mouse Skeletal Muscle.

We investigated the protective effect of Brazilian propolis, a natural resinous substance produced by honeybees, against glycation stress in mouse skeletal muscles. Mice were divided into four groups: (1) Normal diet + drinking water, (2) Brazilian propolis (0.1%)-containing diet + drinking water, (3) normal diet + methylglyoxal (MGO) (0.1%)-containing drinking water, and (4) Brazilian propolis (0.1%)-containing diet + MGO (0.1%)-containing drinking water. MGO treatment for 20 weeks reduced the weight of the extensor digitorum longus (EDL) muscle and tended to be in the soleus muscle. Ingestion of Brazilian propolis showed no effect on this change in EDL muscles but tended to increase the weight of the soleus muscles regardless of MGO treatment. In EDL muscles, Brazilian propolis ingestion suppressed the accumulation of MGO-derived advanced glycation end products (AGEs) in MGO-treated mice. The activity of glyoxalase 1 was not affected by MGO, but was enhanced by Brazilian propolis in EDL muscles. MGO treatment increased mRNA expression of inflammation-related molecules, interleukin (IL)-1ß, IL-6, and toll-like receptor 4 (TLR4). Brazilian propolis ingestion suppressed these increases. MGO and/or propolis exerted no effect on the accumulation of AGEs, glyoxalase 1 activity, and inflammatory responses in soleus muscles. These results suggest that Brazilian propolis exerts a protective effect against glycation stress by inhibiting the accumulation of AGEs, promoting MGO detoxification, and reducing proinflammatory responses in the skeletal muscle. However, these anti-glycation effects does not lead to prevent glycation-induced muscle mass reduction.

Lactate Stimulates a Potential for Hypertrophy and Regeneration of Mouse Skeletal Muscle.

The effects of lactate on muscle mass and regeneration were investigated using mouse skeletal muscle tissue and cultured C2C12 cells. Male C57BL/6J mice were randomly divided into (1) control, (2) lactate (1 mol/L in distilled water, 8.9 mL/g body weight)-administered, (3) cardio toxin (CTX)-injected (CX), and (4) lactate-administered after CTX-injection (LX) groups. CTX was injected into right tibialis anterior (TA) muscle before the oral administration of sodium lactate (five days/week for two weeks) to the mice. Oral lactate administration increased the muscle weight and fiber cross-sectional area, and the population of Pax7-positive nuclei in mouse TA skeletal muscle. Oral administration of lactate also facilitated the recovery process of CTX-associated injured mouse TA muscle mass accompanied with a transient increase in the population of Pax7-positive nuclei. Mouse myoblast-derived C2C12 cells were differentiated for five days to form myotubes with or without lactate administration. C2C12 myotube formation with an increase in protein content, fiber diameter, length, and myo-nuclei was stimulated by lactate. These observations suggest that lactate may be a potential molecule to stimulate muscle hypertrophy and regeneration of mouse skeletal muscle via the activation of muscle satellite cells.

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination.

Recently, it was reported that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-ß-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-ß-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-ß-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-ß-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-ß-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle.

This study investigated the effects of AdipoRon, which is an agonist for adiponectin receptor 1 (AdipoR1) and AdipoR2, on the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells and skeletal muscle mass in C57BL/6J mice. AdipoRon suppressed the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells of C2C12 myotubes in a dose-dependent manner. Adiponectin-associated decline of protein content, diameter, and number of nuclei per myotube in C2C12 myotubes was partially rescued by knockdown of AdipoR1 and/or AdipoR2. Phosphorylation level of AMPK showed a trend to be increased by AdipoRon. A significant increase in phosphorylation level of AMPK was observed at 20 µM AdipoRon. Knockdown of AdipoR1 and/or AdipoR2 rescued AdipoRon-associated decrease in protein content of C2C12 myotubes. AdipoRon-associated increase in phosphorylation level of AMPK in C2C12 myotubes was suppressed by knockdown of AdipoR1 and/or AdipoR2. Successive intravenous injections of AdipoRon into mice caused a decrease in the wet weight of plantaris muscle (PLA), but not in soleus muscle (SOL). Mean fiber cross-sectional area of PLA, but not of SOL, was significantly decreased by AdipoRon administration. On the one hand, the expression level of phosphorylated AMPK and ubiquitinated protein in SOL and PLA muscles was upregulated by AdipoRon administration. On the other hand, AdipoRon administration induced no changes in the expression level of puromycin-labeled proteins in both SOL and PLA muscles. Expression level of adiponectin in extensor digitorum longus (EDL) muscle was increased by aging, but not in SOL muscle. Aging had no effect on the expression level of AdipoR1 and AdipoR2 in both muscles. Phosphorylation level of AMPK in EDL was increased by aging, but not SOL muscle. Results from this study suggest that high level of circulating adiponectin may induce skeletal muscle atrophy, especially fast-type muscle.

AMPK Mediates Muscle Mass Change But Not the Transition of Myosin Heavy Chain Isoforms during Unloading and Reloading of Skeletal Muscles in Mice.

5'AMP-activated protein kinase (AMPK) plays an important role in the regulation of skeletal muscle mass and fiber-type distribution. However, it is unclear whether AMPK is involved in muscle mass change or transition of myosin heavy chain (MyHC) isoforms in response to unloading or increased loading. Here, we checked whether AMPK controls muscle mass change and transition of MyHC isoforms during unloading and reloading using mice expressing a skeletal-muscle-specific dominant-negative AMPKα1 (AMPK-DN). Fourteen days of hindlimb unloading reduced the soleus muscle weight in wild-type and AMPK-DN mice, but reduction in the muscle mass was partly attenuated in AMPK-DN mice. There was no difference in the regrown muscle weight between the mice after 7 days of reloading, and there was concomitantly reduced AMPKα2 activity, however it was higher in AMPK-DN mice after 14 days reloading. No difference was observed between the mice in relation to the levels of slow-type MyHC I, fast-type MyHC IIa/x, and MyHC IIb isoforms following unloading and reloading. The levels of 72-kDa heat-shock protein, which preserves muscle mass, increased in AMPK-DN-mice. Our results indicate that AMPK mediates the progress of atrophy during unloading and regrowth of atrophied muscles following reloading, but it does not influence the transition of MyHC isoforms.

Potential involvement of dietary advanced glycation end products in impairment of skeletal muscle growth and muscle contractile function in mice.

Diets enriched with advanced glycation end products (AGE) have recently been related to muscle dysfunction processes. However, it remains unclear whether long-term exposure to an AGE-enriched diet impacts physiological characteristics of skeletal muscles. Therefore, we explored the differences in skeletal muscle mass, contractile function and molecular responses between mice receiving a diet high in AGE (H-AGE) and low in AGE (L-AGE) for 16 weeks. There were no significant differences between L-AGE and H-AGE mice with regard to body weight, food intake or epididymal fat pad weight. However, extensor digitorum longus (EDL) and plantaris (PLA) muscle weights in H-AGE mice were lower compared with L-AGE mice. Higher levels of N ε -(carboxymethyl)-l-lysine, a marker for AGE, in EDL muscles of H-AGE mice were observed compared with L-AGE mice. H-AGE mice showed lower muscle strength and endurance in vivo and lower muscle force production of PLA muscle in vitro. mRNA expression levels of myogenic factors including myogenic factor 5 and myogenic differentiation in EDL muscle were lower in H-AGE mice compared with L-AGE mice. The phosphorylation status of 70-kDa ribosomal protein S6 kinase Thr389, an indicator of protein synthesis signalling, was lower in EDL muscle of H-AGE mice than that of L-AGE mice. These findings suggest that long-term exposure to an AGE-enriched diet impairs skeletal muscle growth and muscle contractile function, and that these muscle dysfunctions may be attributed to the inhibition of myogenic potential and protein synthesis.

Suppression of Myostatin Stimulates Regenerative Potential of Injured Antigravitational Soleus Muscle in Mice under Unloading Condition.

Effects of myostatin (MSTN)-suppression on the regeneration of injured skeletal muscle under unloading condition were investigated by using transgenic mice expressing a dominant-negative form of MSTN (MSTN-DN). Both MSTN-DN and wild-type (WT) mice were subjected to continuous hindlimb suspension (HS) for 6 weeks. Cardiotoxin (CTX) was injected into left soleus muscle under anesthesia 2 weeks after the initiation of HS. Then, the soleus muscles were excised following 6-week HS (4 weeks after CTX-injection). CTX-injection caused to reduce the soleus fiber cross-sectional area (CSA) in WT mice under both unloading and weight-bearing conditions, but not in MSTN-DN mice. Under unloading condition, CTX-injected muscle weight and fiber CSA in MSTN-DN mice were significantly higher than those in WT mice. CTX-injected muscle had many damaged and regenerating fibers having central nuclei in both WT and MSTN-DN mice. Significant increase in the population of Pax7-positive nuclei in CTX-injected muscle was observed in MSTN-DN mice, but not in WT mice. Evidences indicate that the suppression of MSTN cause to increase the regenerative potential of injured soleus muscle via the increase in the population of muscle satellite cells regardless of unloading conditions.

Involvement of AMPK in regulating slow-twitch muscle atrophy during hindlimb unloading in mice.

AMPK is considered to have a role in regulating skeletal muscle mass. However, there are no studies investigating the function of AMPK in modulating skeletal muscle mass during atrophic conditions. In the present study, we investigated the difference in unloading-associated muscle atrophy and molecular functions in response to 2-wk hindlimb suspension between transgenic mice overexpressing the dominant-negative mutant of AMPK (AMPK-DN) and their wild-type (WT) littermates. Male WT (n = 24) and AMPK-DN (n = 24) mice were randomly divided into two groups: an untreated preexperimental control group (n = 12 in each group) and an unloading (n = 12 in each group) group. The relative soleus muscle weight and fiber cross-sectional area to body weight were decreased by ∼30% in WT mice by hindlimb unloading and by ∼20% in AMPK-DN mice. There were no changes in puromycin-labeled protein or Akt/70-kDa ribosomal S6 kinase signaling, the indicators of protein synthesis. The expressions of ubiquitinated proteins and muscle RING finger 1 mRNA and protein, markers of the ubiquitin-proteasome system, were increased by hindlimb unloading in WT mice but not in AMPK-DN mice. The expressions of molecules related to the protein degradation system, phosphorylated forkhead box class O3a, inhibitor of κBα, microRNA (miR)-1, and miR-23a, were decreased only in WT mice in response to hindlimb unloading, and 72-kDa heat shock protein expression was higher in AMPK-DN mice than in WT mice. These results imply that AMPK partially regulates unloading-induced atrophy of slow-twitch muscle possibly through modulation of the protein degradation system, especially the ubiquitin-proteasome system.

Microcurrent electrical neuromuscular stimulation facilitates regeneration of injured skeletal muscle in mice.

Conservative therapies, mainly resting care for the damaged muscle, are generally used as a treatment for skeletal muscle injuries (such as muscle fragmentation). Several past studies reported that microcurrent electrical neuromuscular stimulation (MENS) facilitates a repair of injured soft tissues and shortens the recovery period. However, the effects of MENS on the regeneration in injured skeletal muscle are still unclear. The purpose of this study was to investigate the effect of MENS on the regenerative process of injured skeletal muscle and to elucidate whether satellite cells in injured skeletal muscle are activated by MENS by using animal models. Male C57BL/6J mice, aged 7 weeks old, were used (n = 30). Mice were randomly divided into two groups: (1) cardiotoxin (CTX)-injected (CX, n = 15) and (2) CTX-injected with MENS treatment (MX, n=15) groups. CTX was injected into tibialis anterior muscle (TA) of mice in CX and MX groups to initiate the necrosis-regeneration cycle of the muscle. TA was dissected 1, 2, and 3 weeks after the injection. Muscle weight, muscle protein content, the mean cross-sectional areas of muscle fibers, the relative percentage of fibers having central nuclei, and the number of muscle satellite cells were evaluated. MENS facilitated the recovery of the muscle dry weight and protein content relative to body weight, and the mean cross-sectional areas of muscle fibers in CTX-induced injured TA muscle. The number of Pax7-positive muscle satellite cells was increased by MENS during the regenerating period. Decrease in the percentages of fibers with central nuclei after CTX-injection was facilitated by MENS. MENS may facilitate the regeneration of injured skeletal muscles by activating the regenerative potential of skeletal muscles. Key pointsMicrocurrent electrical neuromuscular stimulation (MENS) facilitated the recovery of the relative muscle dry weight, the relative muscle protein content, and the mean cross-sectional areas of muscle fibers of injured TA muscle in mice.The number of satellite cells was increased by MENS during the regenerating phase of injured skeletal muscle.Decrease in the percentages of fibers with central nuclei was facilitated by MENS.MENS may facilitate the regeneration of injured skeletal muscles.

Loading-associated expression of TRIM72 and caveolin-3 in antigravitational soleus muscle in mice.

Effects of mechanical loading on the expression level of tripartite motif-containing 72 (TRIM72) and caveolin-3 (Cav-3) in mouse soleus muscle were investigated. Mice were subjected to (1) continuous hindlimb suspension (HS) for 2 weeks followed by 1-week ambulation recovery or (2) functional overloading (FO) on the soleus by cutting the distal tendons of the plantaris and gastrocnemius muscles. Soleus muscle atrophy was induced by 2-week hindlimb suspension (HS). Reloading-associated regrowth of atrophied soleus muscle was observed by 1-week reloading following HS. HS also depressed the expression level of insulin receptor substrate-1 (IRS-1) mRNA, TRIM72, Cav-3, and phosphorylated Akt (p-Akt)/total Akt (t-Akt), but increased the phosphorylated level of p38 mitogen-activated protein kinase (p-p38MAPK) in soleus muscle. Thereafter, the expression level of MyoD mRNA, TRIM72 (mRNA, and protein), and Cav-3 was significantly increased and recovered to the basal level during 1-week reloading after HS. Although IRS-1 expression was also upregulated by reloading, the expression level was significantly lower than that before HS. Significant increase in p-Akt and phosphorylated p70 S6 kinase (p-p70S6K) was observed by 1-day reloading. On the other hand, 1-week functional overloading (FO) induced soleus muscle hypertrophy. In FO-associated hypertrophied soleus muscle, the expression level of IRS-1 mRNA, MyoD mRNA, TRIM72 mRNA, p-Akt, and p-p70S6K was increased, but the expression of Cav-3 and p-p38MAPK was decreased. FO had no effect on the protein expression level of TRIM72. These observations suggest that the loading-associated upregulation of TRIM72 protein in skeletal muscle may depress the regrowth of atrophied muscle via a partial suppression of IRS-1. In addition, downregulation of Cav-3 in skeletal muscle may depress overloading-induced muscle hypertrophy.

AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells.

5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

Up-regulation of adiponectin expression in antigravitational soleus muscle in response to unloading followed by reloading, and functional overloading in mice.

The purpose of this study was to investigate the expression level of adiponectin and its related molecules in hypertrophied and atrophied skeletal muscle in mice. The expression was also evaluated in C2C12 myoblasts and myotubes. Both mRNA and protein expression of adiponectin, mRNA expression of adiponectin receptor (AdipoR) 1 and AdipoR2, and protein expression of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) were observed in C2C12 myoblasts. The expression levels of these molecules in myotubes were higher than those in myoblasts. The expression of adiponectin-related molecules in soleus muscle was observed at mRNA (adiponectin, AdipoR1, AdipoR2) and protein (adiponectin, APPL1) levels. The protein expression levels of adiponectin and APPL1 were up-regulated by 3 weeks of functional overloading. Down-regulation of AdipoR1 mRNA, but not AdipoR2 mRNA, was observed in atrophied soleus muscle. The expression of adiponectin protein, AdipoR1 mRNA, and APPL1 protein was up-regulated during regrowth of unloading-associated atrophied soleus muscle. Mechanical loading, which could increase skeletal muscle mass, might be a useful stimulus for the up-regulations of adiponectin and its related molecules in skeletal muscle.

Regeneration of injured skeletal muscle in heat shock transcription factor 1-null mice.

The purpose of this study was to investigate a role of heat shock transcription factor 1 (HSF1)-mediated stress response during regeneration of injured soleus muscle by using HSF1-null mice. Cardiotoxin (CTX) was injected into the left muscle of male HSF1-null and wild-type mice under anesthesia with intraperitoneal injection of pentobarbital sodium. Injection of physiological saline was also performed into the right muscle. Soleus muscles were dissected bilaterally 2 and 4 weeks after the injection. The relative weight and fiber cross-sectional area in CTX-injected muscles of HSF1-null, not of wild-type, mice were less than controls with injection of physiological saline 4 weeks after the injury, indicating a slower regeneration. Injury-related increase of Pax7-positive muscle satellite cells in HSF1-null mice was inhibited versus wild-type mice. HSF1-deficiency generally caused decreases in the basal expression levels of heat shock proteins (HSPs). But the mRNA expression levels of HSP25 and HSP90α in HSF1-null mice were enhanced in response to CTX-injection, compared with wild-type mice. Significant up-regulations of proinflammatory cytokines, such as interleukin (IL) -6, IL-1ß, and tumor necrosis factor mRNAs, with greater magnitude than in wild-type mice were observed in HSF1-deficient mouse muscle. HSF1 and/or HSF1-mediated stress response may play a key role in the regenerating process of injured skeletal muscle. HSF1 deficiency may depress the regenerating process of injured skeletal muscle via the partial depression of increase in Pax7-positive satellite cells. HSF1-deficiency-associated partial depression of skeletal muscle regeneration might also be attributed to up-regulation of proinflammatory cytokines.

Heat shock transcription factor 1-deficiency attenuates overloading-associated hypertrophy of mouse soleus muscle.

Hypertrophic stimuli, such as mechanical stress and overloading, induce stress response, which is mediated by heat shock transcription factor 1 (HSF1), and up-regulate heat shock proteins (HSPs) in mammalian skeletal muscles. Therefore, HSF1-associated stress response may play a key role in loading-associated skeletal muscle hypertrophy. The purpose of this study was to investigate the effects of HSF1-deficiency on skeletal muscle hypertrophy caused by overloading. Functional overloading on the left soleus was performed by cutting the distal tendons of gastrocnemius and plantaris muscles for 4 weeks. The right muscle served as the control. Soleus muscles from both hindlimbs were dissected 2 and 4 weeks after the operation. Hypertrophy of soleus muscle in HSF1-null mice was partially inhibited, compared with that in wild-type (C57BL/6J) mice. Absence of HSF1 partially attenuated the increase of muscle wet weight and fiber cross-sectional area of overloaded soleus muscle. Population of Pax7-positive muscle satellite cells in HSF1-null mice was significantly less than that in wild-type mice following 2 weeks of overloading (p<0.05). Significant up-regulations of interleukin (IL)-1ß and tumor necrosis factor mRNAs were observed in HSF1-null, but not in wild-type, mice following 2 weeks of overloading. Overloading-related increases of IL-6 and AFT3 mRNA expressions seen after 2 weeks of overloading tended to decrease after 4 weeks in both types of mice. In HSF1-null mice, however, the significant overloading-related increase in the expression of IL-6, not ATF3, mRNA was noted even at 4th week. Inhibition of muscle hypertrophy might be attributed to the greater and prolonged enhancement of IL-6 expression. HSF1 and/or HSF1-mediated stress response may, in part, play a key role in loading-induced skeletal muscle hypertrophy.

Microcurrent electrical nerve stimulation facilitates regrowth of mouse soleus muscle.

Microcurrent electrical nerve stimulation (MENS) has been used to facilitate recovery from skeletal muscle injury. However, the effects of MENS on unloading-associated atrophied skeletal muscle remain unclear. Effects of MENS on the regrowing process of unloading-associated atrophied skeletal muscle were investigated. Male C57BL/6J mice (10-week old) were randomly assigned to untreated normal recovery (C) and MENS-treated (M) groups. Mice of both groups are subjected to continuous hindlimb suspension (HS) for 2 weeks followed by 7 days of ambulation recovery. Mice in M group were treated with MENS for 60 min 1, 3, and 5 days following HS, respectively, under anesthesia. The intensity, the frequency, and the pulse width of MENS were set at 10 µA, 0.3 Hz, and 250 msec, respectively. Soleus muscles were dissected before and immediately after, 1, 3 and 7 days after HS. Soleus muscle wet weight and protein content were decreased by HS. The regrowth of atrophied soleus muscle in M group was faster than that in C group. Decrease in the reloading-induced necrosis of atrophied soleus was facilitated by MENS. Significant increases in phosphorylated levels of p70 S6 kinase and protein kinase B (Akt) in M group were observed, compared with C group. These observations are consistent with that MENS facilitated regrowth of atrophied soleus muscle. MENS may be a potential extracellular stimulus to activate the intracellular signals involved in protein synthesis.