Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca²⁺ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca²⁺ concentration ([Ca²⁺]_i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of 500 nM. Although 70 nM [Ca²⁺]_i does not activate calcineurin-NFAT, we show by the use of Ca²⁺ ionophore that the system is fully activated when [Ca²⁺]_i is 150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca²⁺ concentration. muscle plasticity; NFATc1; resting calcium concentration     

Morphological changes during fiber type transitions in low-frequency-stimulated rat fast-twitch muscle

This study investigates morphological adaptations of rat extensor digitorum longus muscle to chronic low-frequency stimulation (10 Hz, 10 h/d, up to 61±7d). During the early stimulation period (2–4 d), increased basophilia and accumulation of RNA were seen predominantly in type-IIB fibers. Putative satellite cell activation, as indicated by ³H-thymidine incorporation, was also evident during this phase. By 12 d, fiber composition remained unaltered, but there was a decrease in the cross-sectional area of the type-IIB fibers. Following 28 d of low-frequency stimulation, the percentage of type-IIB fibers decreased from 43±3% to 0%, while type-IID fibers increased from 30±3% to 60±6%. The fraction of type-IIA fibers tended to increase (controls 19±3%; stimulated 29±4%), whereas that of the type-I fibers was unaltered (4±1%). At this time, the cross-sectional area of type-IID fibers was unaltered, but that of type-IIA and type-I fibers increased. Further stimulation resulted in a return of type-IID fibers to control levels (23±5%), and a marked increase in type-IIA fibers (45±8%). The percentage of type-I fibers increased from 4±1% to 8±1%. Throughout each stage of chronic stimulation, there was no histological evidence of fiber degeneration and regeneration. These results indicate that, in contrast to the rabbit, chronic low-frequency stimulation-induced fiber conversion in the rat extensor digitorum longus muscle is entirely due to fiber transformation.     

不同训练方式对大鼠骨骼肌纤维类型转化和CaMK II/MEF2传导途径的影响*

目的: 探讨间歇速度训练和耐力训练对大鼠骨骼肌纤维类型转化及钙调蛋白激酶/肌细胞增强因子2(CaMK II/MEF2)信号传导通路的影响。方法: 成年雄性SD大鼠(8周龄)18只,随机分成间歇速度训练组(IST),耐力训练组(ET),设空白组(C),每组6只,IST组采用75 m/min×1 min、20 m/min×1 min的交替训练6次,跑台坡度15,持续时间12 min/d;ET组采用速度30 m/ min、跑台坡度7、持续时间90 min/d的耐力训练。干预8周后,分别取小鼠右侧胫骨前肌、比目鱼肌,酶联免疫吸附法检测骨骼肌琥珀酸脱氢酶(SDH)、乳酸脱氢酶(LDH)活性,ATP酶染色法观察I、Ⅱ型肌纤维面密度、数密度变化情况,SDS-PAGE凝胶电泳技术观察骨骼肌MHC亚型百分比含量、骨骼肌mRNA表达谱测序分析及qRT-PCR技术检测CaN、CaMKII、MEF2水平。结果: 相比C组,ET组SDH活性显著上升,LDH活性降低(P＜0.05);IST组胫骨前肌中LDH活性值升高(P＜0.01)。ET组胫骨前肌MHCIIa%升高,MHCIIb%降低(P＜0.05),比目鱼肌MHCI% 和MHCIIa%升高,MHCIIb%均降低(P＜0.05)。相比IST组,ET组胫骨前肌MHCIIx%升高(P＜0.05)。相比C组,ET组胫骨前肌 I、II 型纤维纤维密度上升,IST组II型纤维纤维密度提高,IST组、ET组比目鱼肌I型纤维纤维密度上升(P＜0.05)。相比C组,ET组骨骼肌CaN、CaMKII、MEF2 mRNA表达水平增高,IST组CaN、CaMKII、MEF2 mRNA表达水平下降(P＜0.01)。Illumina高通量测序筛选骨骼肌纤维转化相关因子及关联分析,运动干预促使骨骼肌纤维转化相关因子表达变化富集于TGF-β/Smad3、CaN/MEF2、AdipoQ等信号通路,而耐力训练显著提高骨骼肌纤维转化、干细胞功能相关信号通路的富集。结论: 耐力训练促进向氧化型肌纤维转化(慢肌),而间歇速度训练向酵解型肌纤维转化(快肌),并伴随着CaMK II/MEF2传导途径中CaN、CaMKII、MEF2基因的高表达。     

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.     

Arginine promotes skeletal muscle fiber type transformation from fast-twitch to slow-twitch via Sirt1/AMPK pathway

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway.     

A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.