Regulation of myosin heavy chain antisense long noncoding RNA in human vastus lateralis in response to exercise training.

Alterations to muscle activity or loading state can induce changes in expression of myosin heavy chain (MHC). For example, sedentary individuals that initiate exercise training can induce a pronounced shift from IIx to IIa MHC. We sought to examine the regulatory response of MHC RNA in human subjects in response to exercise training. In particular, we examined how natural antisense RNA transcripts (NATs) are regulated throughout the MHC gene locus that includes MYH2 (IIa), MYH1 (IIx), MYH4 (IIb), and MYH8 (Neonatal) in vastus lateralis before and after a 5-wk training regime that consisted of a combination of aerobic and resistance types of exercise. The exercise program induced a IIx to IIa MHC shift that was associated with a corresponding increase in transcription on the antisense strand of the IIx MHC gene and a decrease in antisense transcription of the IIa MHC gene, suggesting an inhibitory mechanism mediated by NATs. We also report that the absence of expression of IIb MHC in human limb muscle is associated with the abundant expression of antisense transcript overlapping the IIb MHC coding gene, which is the opposite expression pattern as compared with that previously observed in rats. The NAT provides a possible regulatory mechanism for the suppressed expression of IIb MHC in humans. These data indicate that NATs may play a regulatory role with regard to the coordinated shifts in MHC gene expression that occur in human muscle in response to exercise training.

Alterations in muscle mass and contractile phenotype in response to unloading models: role of transcriptional/pretranslational mechanisms.

Skeletal muscle is the largest organ system in mammalian organisms providing postural control and movement patterns of varying intensity. Through evolution, skeletal muscle fibers have evolved into three phenotype clusters defined as a motor unit which consists of all muscle fibers innervated by a single motoneuron linking varying numbers of fibers of similar phenotype. This fundamental organization of the motor unit reflects the fact that there is a remarkable interdependence of gene regulation between the motoneurons and the muscle mainly via activity-dependent mechanisms. These fiber types can be classified via the primary type of myosin heavy chain (MHC) gene expressed in the motor unit. Four MHC gene encoded proteins have been identified in striated muscle: slow type I MHC and three fast MHC types, IIa, IIx, and IIb. These MHCs dictate the intrinsic contraction speed of the myofiber with the type I generating the slowest and IIb the fastest contractile speed. Over the last ~35 years, a large body of knowledge suggests that altered loading state cause both fiber atrophy/wasting and a slow to fast shift in the contractile phenotype in the target muscle(s). Hence, this review will examine findings from three different animal models of unloading: (1) space flight (SF), i.e., microgravity; (2) hindlimb suspension (HS), a procedure that chronically eliminates weight bearing of the lower limbs; and (3) spinal cord isolation (SI), a surgical procedure that eliminates neural activation of the motoneurons and associated muscles while maintaining neurotrophic motoneuron-muscle connectivity. The collective findings demonstrate: (1) all three models show a similar pattern of fiber atrophy with differences mainly in the magnitude and kinetics of alteration; (2) transcriptional/pretranslational processes play a major role in both the atrophy process and phenotype shifts; and (3) signaling pathways impacting these alterations appear to be similar in each of the models investigated.

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

Calcineurin plays a modulatory role in loading-induced regulation of type I myosin heavy chain gene expression in slow skeletal muscle.

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine (T(3))] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + T(3) was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg x kg(-1) x day(-1)) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + T(3) (P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression.

Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading.

Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

IIx myosin heavy chain promoter regulation cannot be characterized in vivo by direct gene transfer.

In skeletal muscle of the adult mammal IIx is a pivotal myosin heavy chain (MHC) isoform that can be either up- or downregulated depending on both the fiber type of the target muscle and the type of external stimulus imposed. Since little is known about promoter elements of the IIx MHC gene that are important for its transcriptional regulation in vivo,the main goal of this study was to characterize IIx MHC promoter activity and identify potential regulatory elements on the IIx MHC promoter. A direct gene transfer approach was used, and this approach involved transfection of promoter-reporter constructs into intact rat soleus and plantaris muscle under control and denervated conditions, as well as hindlimb suspension (i.e., models to upregulate IIx MHC transcription). Fast-twitch (plantaris) muscle fibers were confirmed to have significantly greater IIx MHC transcriptional products (pre-mRNA and mRNA) than slow-twitch (soleus) muscle fibers. However, promoter sequences corresponding to -2671 to +1720, -1000 to +392, and -605/+392 relative to the IIx MHC transcription start site, plus an additional construct ligated to a putative embryonic MHC enhancer, failed to produce a fiber type-specific response that is characteristic of the endogenous IIx MHC promoter. Furthermore, the activity of these promoter constructs did not demonstrate the expected response to denervation or hindlimb suspension (i.e., marked upregulation), despite normal uptake and activity of a coinjected alpha-actin reference promoter. On the basis of these findings with IIx MHC promoter-reporters we conclude that the loss of the native chromatin environment as well as other necessary cis elements may preclude use of the gene transfer approach, thereby suggesting that there are hidden layers of regulation for the IIx MHC gene.

Dynamics of myosin heavy chain gene regulation in slow skeletal muscle: role of natural antisense RNA.

The evolutionarily conserved order of the skeletal muscle myosin heavy chain (MHC) genes and their close tandem proximity on the same chromosome are intriguing and may be important for their coordinated regulation. We investigated type II MHC gene regulation in slow-type muscle fibers undergoing a slow to fast MHC transformation in response to inactivity, 7 days after spinal cord isolation (SI) in rats. We examined the transcriptional products of both the sense and antisense strands across the IIa-IIx-IIb MHC gene locus. A strand-specific reverse transcription (RT)-PCR approach was utilized to study the expression of the mRNA, the primary transcript (pre-mRNA), the antisense RNA overlapping the MHC genes, and both the intergenic sense and antisense RNAs. Results showed that the mRNA and pre-mRNA of each MHC had a similar response to SI, suggesting regulation of these genes at the transcriptional level. In addition, we detected previously unknown antisense strand transcription that produced natural antisense transcripts (NATs). RT-PCR mapping of the RNA products revealed that the antisense activity resulted in the formation of three major products: aII, xII, and bII NATs (antisense products of the IIa, IIx, and IIb genes, respectively). The aII NAT begins in the IIa-IIx intergenic region in close proximity to the IIx promoter, extends across the 27-kb IIa MHC gene, and continues to the IIa MHC gene promoter. The expression of the aII NAT was significantly up-regulated in muscles after SI, was negatively correlated with IIa MHC gene expression, and was positively correlated with IIx MHC gene expression. The exact role of the aII NAT is not clear; however, it is consistent with the inhibition of IIa MHC gene transcription. In conclusion, NATs may mediate cross-talk between adjacent genes, which may be essential to the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts.

Reliability assessment of ballistic jump squats and bench throws.

The purpose of this investigation was to determine the test-retest reliability and coefficient of variation of 2 novel physical performance tests. Ten healthy men (22.0 +/- 3.0 years, 87.0 +/- 8.0 kg, 20.0 +/- 5.0% body fat) performed 30 continuous and dynamic jump squats (JS) and bench throws (BT) on 4 separate occasions. The movements were performed under loaded conditions utilizing 30% of subject's predetermined 1 repetition maximum in the back squat and bench press. Mean power (MP; W), peak power (PP; W), mean velocity (MV; m.s(-1)), peak velocity (PV; m.s(-1)), and total work (TW; J) were assessed using a ballistic measurement system (Innervations Inc., Muncie, IN). Data were analyzed using repeated measures analysis of variance with Duncan's post hoc test when mean differences were p < or = 0.05. Intraclass correlation coefficient (ICC) and within-subject coefficient of variation (CV%) were also calculated. All values are presented as mean +/- SE. BT variables were statistically similar across the 4 sessions: MP (350.0 +/- 13.9 W), PP (431.4 +/- 18.5 W) MV (1.6 +/- 0.03 m.s(-1)), PV (2.0 +/- 0.03 m.s(-1)), and TW (199.1 +/- 7.2 J). For JS, session 3 PP (1,669.8 +/- 111.2 W) was significantly greater vs. sessions 1, 2, and 4 (1,601.2 +/- 58.4 W). Session 4 MP (1,403.2 +/- 88.6 W) and MV (1.9 +/- 0.1 m.s(-1)) for JS were significantly lower during sessions 1, 2, and 3 (MP: 1,479.4.5 +/- 44.8 W, MV: 2.0 +/- 0.05 m.s(-1)). TW (834.7 +/- 24.3 J) and PV (2.2 +/- 0.04 m.s(-1)) were statistically similar during all sessions for JS. The CVs ranged from 3.0 to 7.6% for the BT and 3.2 to 5.7% for the JS. ICCs for MP, PP, MV, PV, and TW were 0.92, 0.95, 0.94, 0.91, and 0.95, respectively, during BT. ICCs during JS for MP, PP, MV, PV, and TW were 0.96, 0.98, 0.94, 0.94, and 0.89, respectively. The results of the current study support the use of a 30 continuous and dynamic BT protocol as a reliable upper-body physical performance test, which can be administered with minimal practice. Slightly greater variability for JS was observed, although the test had high reliability.

IGF-I system responses during 12 weeks of resistance training in end-stage renal disease patients.

OBJECTIVE: To examine the hypothesis that 12 weeks of resistance training would alter circulating concentrations of IGF-I system components in end-stage renal disease (ESRD) patients. DESIGN: Ten ESRD patients underwent 12 weeks of resistance training after a 6 week control period and had morning fasted blood drawn on four occasions (weeks - 6, 0, 6, 12). Immunoassays were performed for serum total and free IGF-I, IGF binding proteins (IGFBPs) 2 and 3, and the acid labile subunit (ALS). Immunoaffinity depletion of ALS-based complexes allowed measurement of non-ternary (i.e., binary) IGF-I and IGFBP-3. RESULTS: Significant improvements in strength and functional performance were observed. All IGF-I measures were stable during the control period and no changes were observed for the first 6 weeks of resistance training. At week 12, total IGF-I (-15.4+/-28.9%), ternary IGF-I (-16.4+/-36.7%), and the IGF-I/IGFBP-3 ratio had significantly (p < or = 0.05) declined from week 0 values. No changes were observed for free IGF-I, IGFBPs 2 and 3, or the acid labile subunit. The proportion of IGF-I in ternary ( approximately 76.3+/-6.8%), non-ternary ( approximately 22.5+/-6.6%), and free ( approximately 1.2+/-0.5%) forms remained constant throughout the training. CONCLUSIONS: 12 weeks of resistance training in ESRD patients induced a decline in total IGF-I, but did not alter the proportion of IGF-I circulating in free, ternary or non-ternary molecular complexes. The decline in IGF-I occurs in the presence of positive training adaptations on physical performance and we conclude that this response pattern appears to be reflective of favorable neuromuscular anabolic adaptations.

Reliability assessment of two militarily relevant occupational physical performance tests.

To determine the number of test sessions needed to stabilize performance on two military occupational physical tests and to assess their reliability, 10 male soldiers (22 +/- 3 yrs, 183 +/- 7 cm, 87 +/- 8 kg) performed both an indoor 6-station obstacle course (OC) and a repetitive box-lifting task (RBLT). The OC consisted of 46 cm-high hurdles, zigzag sprint, low crawl, horizontal pipe shimmy, 1.4 m wall traversal, and straight sprint. The RBLT required subjects to lift 20.5 kg boxes, continuously for 10 minutes, from the ground onto 1.3 m high platforms positioned 2.4 m apart. The OC mean +/- SD times (s), for sessions 1-4 respectively, were 37.4 +/- 2.2, 35.8 +/- 2.5, 34.7 +/- 2.1, and 34.5 +/- 1.7 seconds. The number of boxes lifted was 177 +/- 31, 194 +/- 28, 189 +/- 32, and 186 +/- 37 for the RBLT. Performance stabilized on the 3rd session for the OC (7% improvement over first trial, p < 0.05) and on the 2nd session for the RBLT (9% improvement over first trial, p < 0.05). The intraclass correlation coefficients were 0.92 and 0.94 for the OC and RBLT, respectively. This study demonstrates that both are reliable tests, but they do require administration of 1 single-trial session of RBLT and 2 two-trial sessions of OC before highly reliable performance data are obtained.

Correlates of load carriage and obstacle course performance among women.

To examine correlates of the speed at which female soldiers carrying loads could cover 3.2 km on foot and traverse an obstacle course, 12 volunteers (mean +/- SD: 25.3 +/- 6 years, 166 +/- 7 cm, 61.3 +/- 7 kg) were timed over 3.2 km while carrying loads of 14, 27, and 41 kg, and while traversing an obstacle course with the two lighter loads. Pearson correlations showed that absolute VO_[2 max] and 3.2 km run time without a load were the best predictors of 3.2 km load carriage time for all loads. Also, larger subjects with greater muscle mass were able to carry the heaviest load faster than smaller, less muscular subjects, likely because the 41 kg load represented a smaller percentage of the former's bodyweight. Maximum number of sit-ups and push-ups, composite score of the Army Physical Fitness Test as well as body height were positively correlated with the speed at which some course segments were traversed.