Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes.

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.

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.

Administration of granulocyte colony-stimulating factor facilitates the regenerative process of injured mice skeletal muscle via the activation of Akt/GSK3alphabeta signals.

Effects of administration of granulocyte colony-stimulating factor (G-CSF) on the regeneration of injured mammalian skeletal muscles were studied in male C57BL/6J mice. Muscle injury was induced by injection of cardiotoxin (CTX) into tibialis anterior muscles bilaterally. G-CSF was administrated for 8 consecutive days from 3 days before and 5 days after the injection. Significant decreases of wet weight and protein content were noted in the necrotic muscle with CTX injection. A large number of the regenerating fibers having central nucleus were observed 7 days after the injection. The regeneration of injured muscle was further facilitated by the G-CSF treatment. Population of Pax7-positive nuclei was increased by the G-CSF treatment at day 7. Phospho-Akt and phospho-glycogen synthase kinase 3alphabeta (GSK3alphabeta) signals were also activated by G-CSF-administrated group during the regenerative process. It was suggested that G-CSF treatment may facilitate the regeneration of injured skeletal muscles via the activation of Akt/GSK3alphabeta signals.

Functional overloading facilitates the regeneration of injured soleus muscles in mice.

The effect of functional overloading on the regenerating process of injured skeletal muscle was investigated in 10-week-old male mice (C57BL/6J). Functional overloading on soleus of both hindlimbs was performed by cutting the distal tendons of plantaris and gastrocnemius muscles for 2 weeks before cardiotoxin (CTX) injection as the preconditioning and also during 10 weeks of recovery. To activate the necrosis-regeneration cycle, 0.1 ml of 10-microM CTX was injected into soleus muscle. The mean values of absolute muscle weight and the percentage of Pax7-positive nuclei in soleus were increased by the preconditioning. These values, as well as total muscle protein content, in the group with CTX injection plus overloading were larger than in the group with CTX injection alone. Fibers with central nucleus were noted in the group with CTX injection with or without overloading. The rate of disappearance of fibers having central nucleus during recovery was stimulated by overloading. Histological analyses revealed that the regeneration of injured soleus muscle with overloading proceeded more rapidly than the muscle without overloading. These results, in combination with previous lines of evidence, strongly suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.

Geranylgeranylaceton induces heat shock protein 72 in skeletal muscle cells.

Effects of an antiulcer drug, geranylgeranylaceton (GGA), and/or heat-stress on 72 kDa heat shock protein (HSP72) expression and protein content in cultured skeletal muscle cells were studied. Mouse skeletal muscle cells (C(2)C(12)) were subjected to either 1) control (cultured at 37 degrees C without GGA), 2) GGA administration (10(-11) - 10(-8) M), 3) heat-stress at 41 degrees C for 60 min, or 4) GGA administration combined with heat-stress. Expression of HSP72 was up-regulated by GGA administration. Heat-stress further enhanced the GGA-related up-regulation of HSP72. Administration of GGA caused an increase of muscular protein content as a dose-dependent manner. Protein synthesis was also stimulated by heat-stress alone in myotubes. It was suggested that GGA stimulates the differentiation of myoblasts and protein synthesis. These observations may also suggest that the administration of GGA could be one of the useful tools to gain muscular mass not only in athletes, but also in patients during rehabilitation.

Effects of functional overloading on the regenerative potential of injured skeletal muscles in mice.

The purpose of this study was to investigate the effects of functional overload on the regeneration of injured skeletal muscles of male C57BL/6J mice. To activate a necrosis-regeneration cycle, cardiotoxin (CTX) was injected into soleus muscles both control and functionally overloaded groups. The recovery of muscle protein content, which was decreased by CTX injection, was significantly stimulated by application of functional overloading. The CTX-injection-related increment of satellite cell number in the overloaded groups was also greater than that in the group without overloading. Evidences suggest that the application of a mechanical stress on the injured skeletal muscles could activate satellite cells and facilitate the regeneration of the muscle.