Skeletal Muscle Fiber Type Research Articles

Tensiomyographic Assessment of Muscle Contractile Properties in 9- to 14-Year Old Children.

While there are numerous data on the skeletal muscle fiber type composition in adults, little is known about the changes in fiber type composition and contractile properties during maturational growth in children. Using noninvasive tensiomyography, we measured contraction time (Tc), an indirect estimate of the myosin heavy chain I (MHC-I) proportion, to assess the longitudinal changes of the biceps brachii (BB), biceps femois (BF), vastus lateralis (VL), and erector spinae (ES) muscles in 53 boys and 54 girls. The children were 9 years at the start of the study and returned for 5 follow-up measurements until the age of 14 years. The ES has the shortest and the BF has the longest Tc. The VL and ES of boys have shorter Tc than those of girls. When applying the relationship between proportion of MHC-I and Tc established in adults to children's TMG data, we found a slow-to-fast transition in the VL between, at least, the ages of 6 to 10 years, when it stabilized to adult proportions. Regular participation in sports was associated with a faster BF, but not in the VL. Our data represents a first non-invasive indication of the developmental changes in muscle fiber type composition in children.

Phytol Promotes the Formation of Slow-Twitch Muscle Fibers through PGC-1α/miRNA but Not Mitochondria Oxidation.

Phytol is a side chain of chlorophyll belonging to the side-chain double terpenoid. When animals consume food rich in chlorophyll, phytol can be broken down to phytanic acid after digestion. It was reported that feeding animals with different varieties and levels of forage could significant improve pH and marbling score of steer and lamb carcasses, but the internal mechanism for this is still not reported. The marbling score and pH of muscle was mainly determined by skeletal muscle fiber type, which is due to expression of different myosin heavy-chain (MHC) isoforms. Here, we provide evidence that phytol can indeed affect the diversity of muscle fiber types both in vitro and in vivo and demonstrate that phytol can increase the expression of MHC I (p < 0.05), likely by upgrading the expression of PPARδ, PGC-1α, and related miRNAs. This fiber-type transformation process may not be caused by activated mitochondrial metabolism but by the structural changes in muscle fiber types.

Effects of aerobic exercise on the expressions of skeletal muscle fiber type-related genes and FNDC-5 in high-fat diet-induced obese rats

Obesity is known to be linked with a shift in skeletal muscle fiber type, which plays a role in insulin sensitivity and myokines activity. Here, we investigated whether aerobic exercise training has any effects on skeletal muscle MHC isoforms related to gene expression, irisin, and FNDC5 in obese rats. Thirty-two male SD-rats (3 wks old) were randomly assigned to normal diet (ND, n = 8) and high-fat diet (HFD, n = 24) groups, with specific caloric intake (ND; 11.5%, HFD; 60% of kcals from fat), for 8 weeks. After 8 weeks, HFD-induced obese rats were randomly reassigned to either an obese control (OC, n = 8) or obese exercise (OE, n = 8) group, and provided a normal diet. The OE group was exercised on the rodent treadmill (60 min, 15 m/min, 5 times) for 8 weeks. We investigated FNDC5, slow-twitch muscle gene (miR-499) and fast-twitch muscle gene (SOX6) expression in gastrocnemius muscle, and serum irisin. HFD group showed heavier body weight compared to ND group (p &lt; .05). Blood glucose concentrations, determined by IGTT, were significantly higher in HFD group compared to ND group in 90-minutes and 120-minutes of the recovery phase (p &lt; .05, 43%, 34%). Average body weight for the OE group was significantly lower than the OC group. The level of blood glucose in the OE group was lower than the OC group in 90-minutes and 120-minutes of the recovery phase (p &lt; .05, 11%, 9%). Irisin levels were not significantly different between the HFD and ND groups. However, FNDC5 level was significantly lower in HFD rats than ND (p &lt; .05, 53%). The OE group showed increased expression of irisin and FNDC5 compared to ND, but there was no change in OC (p &lt; .05, 23%, 42%). While the levels of miR-499 and SOX6 were significantly higher in the OE compared to OC group, there was no change in MYH7. Taken together, 8 week regular aerobic exercise has positive effects on body weight, blood irisin concentration, its precursor FNDC5, as well as fiber type promoting genes mi-499 and SOX6.

Associations of human skeletal muscle fiber type and insulin sensitivity, blood lipids, and vascular hemodynamics in a cohort of premenopausal women.

Cardiometabolic disease remains a leading cause of morbidity and mortality in developed nations. Consequently, identifying and understanding factors associated with underlying pathophysiological processes leading to chronic cardio metabolic conditions is critical. Metabolic health, arterial elasticity, and insulin sensitivity (SI) may impact disease risk, and may be determined in part by myofiber type. Therefore, the purpose of this study was to test the hypothesis that type I myofiber composition would be associated with high SI, greater arterial elasticity, lower blood pressure, and blood lipids; whereas, type IIx myofibers would be associated with lower SI, lower arterial elasticity, higher blood pressure, blood lipids. Muscle biopsies were performed on the vastus lateralis in 16 subjects (BMI=27.62±4.71kg/m2, age=32.24±6.37years, 43% African American). The distribution of type I, IIa, and IIx myofibers was determined via immunohistochemistry performed on frozen cross-sections. Pearson correlation analyses were performed to assess associations between myofiber composition, SI, arterial elasticity, blood pressure, and blood lipid concentrations. The percentage of type I myofibers positively correlated with SI and negatively correlated with systolic blood pressure SBP, diastolic blood pressure, and mean arterial pressure (MAP); whereas, the percentage of type IIx myofibers were negatively correlated with SI and large artery elasticity, and positively correlated with LDL cholesterol, SBP, and MAP. These data demonstrate a potential link between myofiber composition and cardiometabolic health outcomes in a cohort of premenopausal women. Future research is needed to determine the precise mechanisms in which myofiber composition impacts the pathophysiology of impaired glucose and lipid metabolism, as well as vascular dysfunction.

Determinants of skeletal muscle work efficiency in patients with COPD

Skeletal muscle work inefficiency plays a major role in limiting exercise capacity in patients with chronic Obstructive Pulmonary Disease (COPD). However, the physiological mechanisms responsible for this excessive energy demand from the exercising muscle of patients with COPD remains poorly understood. To address this gap in our knowledge, we measured the cost of muscle contraction (ATP cost) in the quadriceps of patients with moderate to severe COPD (n=11) and age‐matched sedentary controls (n=12) with phosphorus magnetic resonance spectroscopy (31P‐MRS) during 1) maximal intermittent contractions to elicit a metabolic demand from both cross‐bridge cycling and ion pumping and 2) a continuous maximal contraction to predominantly tax cross‐bridge cycling. In addition, muscle fiber type distribution was determined in the vastus lateralis using immunofluorescence analysis. The ATP cost of contraction for both the continuous (Control: 0.11 ± 0.07 mM.min−1.N m−1; COPD: 0.12 ± 0.07 mM.min−1.N m−1) and intermittent (Control: 0.24 ± 0.17 mM.min−1.N m−1; COPD: 0.24 ± 0.18 mM.min−1.N m−1) protocols were not significantly different between groups with or without normalization for muscle mass (P&gt;0.05). Somewhat surprisingly, fiber type distribution was also not different between groups (Type I; Control: 41 ± 12 %; COPD: 40 ± 18 %; Type II; Control: 59 ± 12 %; COPD: 60 ± 18 % respectively P&gt;0.05). Of note, this finding of similar fiber type composition in controls and COPD was partly the consequence of a relatively high proportion of type II fibers in the elderly controls. These, somewhat serendipitous, data reveal that excessive energy demand from ion pumping or myosin ATPase per se is not an obligatory component of the skeletal muscle work inefficiency in COPD and suggest that the previously documented elevated ATP cost of contraction in the locomotor muscles of these patients was likely mediated by a fiber type shift. In practical terms, these findings highlight the potential importance of developing effective therapeutic strategies to limit the likely deleterious consequences of a skeletal muscle fiber type shift in the lower limbs of patients with COPD.Support or Funding InformationThis work was funded in part by grants from the Flight Attendant Medical Research Institute (FAMRI), NIH National Heart, Lung, and Blood Institute (PO1 HL 091830, K99HL125756) and VA Merit grant E6910R.

1,25‐Dihydroxyvitamin D3 Mediates L6 Myoblast Differentiation via Vitamin D Receptor (VDR)

In our previous study, it was demonstrated that skeletal muscle fiber type composition was changed by dietary supplementation of 25‐hydroxyvitamin D3 in growing rats. Potential mechanisms of 25‐hydroxyvitamin D3, a precursor of 1,25‐dihydroxyvitamin D3 (active form), have been suggested in previous studies that VDR was involved with mouse C2C12 myoblasts. However, it has not been explained in rat L6 myoblasts. In this study, we examined the role of VDR in skeletal muscle differentiation and fiber type‐specific gene expression by using short hairpin RNA (shRNA) for VDR knockdown in rat L6 myoblast. L6 myoblasts transfected either with scrambled or shVDR vector were treated with vehicle (CON), 1 pM, 10 pM, or 100 pM 1,25‐dihydroxyvitamin D3 (VitD) for 8 days of differentiation. VDR knockdown was confirmed by Western blot. And myogenic regulatory markers and fiber type‐specific gene expression levels were determined by qPCR. VDR protein was increased by VitD at 10 pM and 100 pM compared to that of CON (1.46 and 1.89 folds, respectively, p&lt;0.05). However, shVDR was effective in suppressing VDR up to 10 pM as VDR protein levels were decreased to 34 %, 24 %, and 39 % (CON, 1 pM, and 10 pM, respectively, p&lt;0.05) compared to scrambled vector. And at 100 pM VitD, VDR was not repressed by shVDR compared to that of scrambled vector. Relative gene expression levels of both MyoD and Myogenin were downregulated by VDR knockdown in CON (0.56 and 0.36 folds, respectively, p&lt;0.05), however, VitD treatment abolished the effects. Fiber type‐specific genes were also modulated by VDR knockdown. Gene expression level of Myh7, dominantly expressed gene in slow‐twitch fiber, was suppressed to 37 %, 29 %, and 43 % (CON, 1 pM, and 10 pM, respectively, p&lt;0.05) by VDR knockdown compared to scrambled vector, but was restored at 100 pM. Myh2, which consists of fast‐twitch fiber, was also downregulated by VDR knockdown at CON (0.57 folds, p&lt;0.05), but its level was restored by VitD treatment. The present study demonstrates that VDR affects mRNA levels of the genes involved in L6 myoblast differentiation and fiber type specificity. Also, the VDR knockdown effect was partially modulated by VitD. It could suggest that the effects of VitD on myogenic differentiation might be partially mediated through VDR.

Transfer of \u03b2-hydroxy-\u03b2-methylbutyrate from sows to their offspring and its impact on muscle fiber type transformation and performance in pigs

BackgroundPrevious studies suggested that supplementation of lactating sows with β-hydroxy-β-methylbutyrate (HMB) could improve the performance of weaning pigs, but there were little information in the muscle fiber type transformation of the offspring and the subsequent performance in pigs from weaning through finishing in response to maternal HMB consumption. The purpose of this study was to determine the effect of supplementing lactating sows with HMB on skeletal muscle fiber type transformation and growth of the offspring during d 28 and 180 after birth. A total of 20 sows according to their body weight were divided into the control (CON, n = 10) or HMB groups (HMB, n = 10). Sows in the HMB group were supplemented with β-hydroxy-β-methylbutyrate calcium (HMB-Ca) 2 g /kg feed during d 1 to 27 of lactation. After weaning, 48 mixed sex piglets were blocked by sow treatment and fed standard diets for post-weaning, growing, finishing periods. Growth performance was recorded during d 28 to 180 after birth. Pigs were slaughtered on d 28 (n = 6/treatment) and 180 (n = 6/treatment) postnatal, and the longissimus dorsi (LD) was collected, respectively.ResultsThe HMB-fed sows during lactation showed increased HMB concentration (P < 0.05) in milk and LD of weaning piglets (P < 0.05). In addition, offsprings in HMB group had a higher finishing BW and lean percentage than did pigs in CON group (P < 0.05), meanwhile, compared with pigs from sows fed the CON diet, pigs from sows fed HMB diet showed higher type II muscle fiber cross-sectional area (CSA), elevated myosin heavy chain (MyHC) IIb and Sox6 mRNA, and fast-MyHC protein levels in LD (P < 0.05).ConclusionsHMB supplemented to sow diets throughout lactation increases the levels of HMB in maternal milk and skeletal muscle of pigs during d 28 after birth and promotes subsequent performance of pigs between d 28 and 180 of age by enhancing glycolytic muscle fiber transformation.

画像処理による骨格筋の筋線維タイプ分別法

画像処理による骨格筋の筋線維タイプ分別法

Mice Hemizygous for a Pathogenic Mitofusin-2 Allele Exhibit Hind Limb/Foot Gait Deficits and Phenotypic Perturbations in Nerve and Muscle.

Charcot-Marie-Tooth disease type 2A (CMT2A), the most common axonal form of hereditary sensory motor neuropathy, is caused by mutations of mitofusin-2 (MFN2). Mitofusin-2 is a GTPase required for fusion of mitochondrial outer membranes, repair of damaged mitochondria, efficient mitochondrial energetics, regulation of mitochondrial-endoplasmic reticulum calcium coupling and axonal transport of mitochondria. We knocked T105M MFN2 preceded by a loxP-flanked STOP sequence into the mouse Rosa26 locus to permit cell type-specific expression of this pathogenic allele. Crossing these mice with nestin-Cre transgenic mice elicited T105M MFN2 expression in neuroectoderm, and resulted in diminished numbers of mitochondria in peripheral nerve axons, an alteration in skeletal muscle fiber type distribution, and a gait abnormality.

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen.

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.

Influence of exercises using different energy metabolism systems on NNMT expression in different types of skeletal muscle fibers

Summary Purpose Nicotinamide N-methyltransferase (NNMT) plays important roles in energy metabolism and in numerous non-infectious chronic diseases (NCD). The aim of this paper is to observe the influence of the different energy metabolism types on the NNMT expression, which is helpful to understand the roles of NNMT in energy metabolism and to develop exercise therapies against NCD. Methods Thirty Sprague-Dawley rats were randomly distributed into three groups, the control group (CG), the below lactate threshold (LT) group (BLTG) and the supra lactate threshold group (SLTG), and then a 6-week run training program was performed. Before the formal training, the blood lactate of the rats was measured immediately after a 30 min continuous running. Twenty-four hours after the last training session, the extensor digitorum longus muscle (EDL) and the soleus muscles were isolated for analysis of western blot. Results The blood lactate level of the BLTG is only slightly higher than that of the CG (P > 0.05), but that of the SLTG is highly significantly higher than that of the CG or of the BLTG (P Conclusion The glycolytic metabolism is significantly more effective to increase the NNMT expression than the aerobic metabolism, and the anaerobic exercise relying on the glycolytic metabolism system could be used to increase the NNMT expression.

FoxO1: a novel insight into its molecular mechanisms in the regulation of skeletal muscle differentiation and fiber type specification.

FoxO1, a member of the forkhead transcription factor forkhead box protein O (FoxO) family, is predominantly expressed in most muscle types. FoxO1 is a key regulator of muscle growth, metabolism, cell proliferation and differentiation. In the past two decades, many researches have indicated that FoxO1 is a negative regulator of skeletal muscle differentiation while contrasting opinions consider that FoxO1 is crucial for myoblast fusion. FoxO1 is expressed much higher in fast twitch fiber enriched muscles than in slow muscles and is also closely related to muscle fiber type specification. In this review, we summarize the molecular mechanisms of FoxO1 in the regulation of skeletal muscle differentiation and fiber type specification.

From neuroleptics to neuroscience and from Pavlov to psychotherapy: more than just the "emperor's new treatments" for mental illnesses?

“The drugs don't work” was one of the hit singles from The Verve's album Urban Hymns , released in 1997. The song was written by lead singer Richard Ashcroft relating to his drug abuse, but it might well relate to the modern treatments for mental illnesses. More than half a century after neuroleptics, antidepressants, benzodiazepines, antipsychotics, behavior therapy, and cognitive treatment were introduced, it is prudent to ask whether “the drugs don't work”. During the past 50 years, the industrialized world has seen a dichotomy between loudly proclaimed therapeutic breakthroughs and rapidly rising numbers of people on disability payments because of mental illness. We hear that antipsychotic, antidepressant, and anti‐anxiety drugs as well as behavior therapy and newer treatments have radically improved what was described as a dismal fate of people suffering from mental disorders. Simultaneously, the percentage of disabled mentally ill patients in the USA has risen by more than 600% since the 1950s (Whitaker, 2010) and similar rates are seen in European countries. Most epidemiologists agree that this “epidemic” is not caused by increased incidence. Moreover, the once rapid succession of new therapeutic developments seems to have halted, at least in pharmacology, as big companies are withdrawing from research on mental disorders. How can this apparent contradiction be explained? Could it be that therapeutic progress is much less than we think or are being told? Could it be that the course of depression, anxiety, schizophrenia, or ADHD has been altered for the worse? Could it be that we cannot make therapeutic progress because the concept of mental illness and its treatment is deeply flawed? There are strong reasons to assume that all three suspicions are in fact true. So, what do we know about the efficiency of pharmacological and psychological treatments? In regard to short‐term outcomes, pharmacotherapy is clearly …

Inferring the Skeletal Muscle Developmental Changes of Grazing and Barn-Fed Goats from Gene Expression Data.

Thirty-six Xiangdong black goats were used to investigate age-related mRNA and protein expression levels of some genes related to skeletal muscle structural proteins, MRFs and MEF2 family, and skeletal muscle fiber type and composition during skeletal muscle growth under grazing (G) and barn-fed (BF) feeding systems. Goats were slaughtered at six time points selected to reflect developmental changes of skeletal muscle during nonrumination (days 0, 7, and 14), transition (day 42), and rumination phases (days 56 and 70). It was observed that the number of type IIx in the longissimus dorsi was increased quickly while numbers of type IIa and IIb decreased slightly, indicating that these genes were coordinated during the rapid growth and development stages of skeletal muscle. No gene expression was affected (P > 0.05) by feeding system except Myf5 and Myf6. Protein expressions of MYOZ3 and MEF2C were affected (P < 0.05) by age, whereas PGC-1α was linearly decreased in the G group, and only MYOZ3 protein was affected (P < 0.001) by feeding system. Moreover, it was found that PGC-1α and MEF2C proteins may interact with each other in promoting muscle growth. The current results indicate that (1) skeletal muscle growth during days 0-70 after birth is mainly myofiber hypertrophy and differentiation, (2) weaning affects the expression of relevant genes of skeletal muscle structural proteins, skeletal muscle growth, and skeletal muscle fiber type and composition, and (3) nutrition or feeding regimen mainly influences the expression of skeletal muscle growth genes.

Structural and physiological muscle changes after post-stroke hemiplegia: A systematic review

Corticospinal tract damage is responsible for motor function impairment after stroke. However, many processes seem to induce muscle damage, which may limit rehabilitation achievement. To achieve a systematic literature review of human muscular modifications due to post-strike hemiplegia. This review considers structural (histological, biochemical) and physiological modifications and their functional consequences over 1 year after stroke. Literature search on PubMed , Embase and Cochrane databases for papers published before February 2016 combining the following keywords cerebral stroke, hemiplegic, atrophy, muscle structure, paresis, skeletal muscle fiber type, motor unit, oxidative stress, strength, motor control led to select forty articles. Literature reports atrophy (20–25%), fatty replacement (17–30,6%), and decrease in fibers size and type (43% of type I fibers) as well as capillary density (38%) in spastic-paretic muscle. Enzymatic activity reduction was observed (decrease in citrate synthase and 3-hydroxyacyl-coenzymeA-deshydrogenase concentrations). A physiological reduction in the motor unit number estimate occurs very early (starting from 4 h post-stoke). At a chronic stage, an increase of motor unit firing rate and motor unit activity at “rest” were reported. These global changes have an additional effect on motor command impairments and increase the motor deficiency, even at a chronic stage. The initial dysfunction of motor command in post-stroke hemiplegia induces structural and physiological muscular changes. These changes are hardly reversible, and plead for an improvement in muscular training in these patients.

Epigallocatechin Gallate Reduces Slow-Twitch Muscle Fiber Formation and Mitochondrial Biosynthesis in C2C12 Cells by Repressing AMPK Activity and PGC-1α Expression.

Epigallocatechin gallate (EGCG) is a major active compound in green tea polyphenols. EGCG acts as an antioxidant to prevent the cell damage caused by free radicals and their derivatives. In skeletal muscle, exercise causes the accumulation of intracellular reactive oxygen species (ROS) and promotes the formation of slow-type muscle fiber. To determine whether EGCG, as a ROS scavenger, has any effect on skeletal muscle fiber type, we applied different concentrations (0, 5, 25, and 50 μM) of EGCG in the culture medium of differentiated C2C12 cells for 2 days. The fiber-type composition, mitochondrial biogenesis-related gene expression, antioxidant and glucose metabolism enzyme activity, and ROS levels in C2C12 cells were then detected. According to our results, 5 μM EGCG significantly decreased the cellular activity of SDH, 25 μM EGCG significantly downregulated the MyHC I, PGC-1α, NRF-1, and p-AMPK levels and SDH activity while enhancing the CAT and GSH-Px activity and decreasing the intracellular ROS levels, and 50 μM EGCG significantly downregulated MyHC I, PGC-1α, and NRF-1 expression and HK and SDH activity while increasing LDH activity. Furthermore, 300 μM H2O2 and 0.5 mM AMPK agonist (AICAR) improved the expression of MyHC I, PGC-1α, and p-AMPK, which were all reversed by 25 μM EGCG. In conclusion, the effect of EGCG on C2C12 cells may occur through the reduction of the ROS level, thereby decreasing both AMPK activity and PGC-1α expression and eventually reducing slow-twitch muscle fiber formation and mitochondrial biosynthesis.

Coupling of mitochondrial function and skeletal muscle fiber type by a miR‐499/Fnip1/ AMPK circuit

Upon adaption of skeletal muscle to physiological and pathophysiological stimuli, muscle fiber type and mitochondrial function are coordinately regulated. Recent studies have identified pathways involved in control of contractile proteins of oxidative‐type fibers. However, the mechanism for coupling of mitochondrial function to the muscle contractile machinery during fiber type transition remains unknown. Here, we show that the expression of the genes encoding type I myosins, Myh7/Myh7b and their intronic miR‐208b/miR‐499, parallels mitochondrial function during fiber type transitions. Using in vivo approaches in mice, we found that miR‐499 drives a PGC‐1α‐dependent mitochondrial oxidative metabolism program to match shifts in slow‐twitch muscle fiber composition. Mechanistically, miR‐499 directly targets Fnip1, an AMP‐activated protein kinase (AMPK)‐interacting protein that negatively regulates AMPK, a known activator of PGC‐1α. Inhibition of Fnip1 reactivated AMPK/PGC‐1α signaling and mitochondrial function in myocytes. Restoration of the expression of miR‐499 in the mdx mouse model of Duchenne muscular dystrophy (DMD) reduced the severity of DMD. Thus, we have identified a miR‐499/Fnip1/AMPK circuit that can serve as a mechanism to couple muscle fiber type and mitochondrial function.

C/EBPα represses slow myosin heavy chain 2 gene expression in developing avian myotubes

BackgroundThe CCAAT/enhancer binding proteins (C/EBP) comprise a family of transcription factors that regulate many cellular processes. Little is known of their function during embryonic and fetal myogenesis. Slow myosin heavy chain 2 (MyHC2) is a marker of the slow avian skeletal muscle fiber type, and slow MyHC2 gene regulation involves molecular pathways that lead to muscle fiber type diversification. MethodsThe biological effects of C/EBPα and C/EBPβ expression were analyzed by use of a general C/EBP activity reporter and by slow MyHC2 promoter-reporter constructs transfected into specific myogenic cell lineages. The effects of C/EBPα and C/EBPβ expression were also analyzed by immunocytochemical detection of slow MyHC2. C/EBPα interaction with the slow MyHC2 promoter was assessed by electromobility shift assays. ResultsC/EBPα and C/EBPβ are present in embryonic fast and fast/slow avian myogenic lineages. Overexpression of C/EBPα cDNA repressed slow MyHC2 promoter activity in embryonic myotubes and in both electrically stimulated fetal myotubes. Deletion analysis of the slow MyHC2 promoter-luciferase reporter demonstrated that the transcriptional repression mediated by C/EBPα occurs within the first 222bp upstream from exon 1 of the slow MyHC2 gene. Electromobility shift assays determined that C/EBPα can bind to a non-canonical C/EBP site within the slow MyHC2 gene, and mutation of this site reduced transcriptional repression of the slow MyHC2 gene. ConclusionC/EBPα, but not C/EBPβ, represses slow MyHC2 promoter activity via a non-canonical C/EBP binding element. General significanceMembers of the C/EBP family of transcription factors differentially regulate genes indicative of distinct muscle fiber types.

Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.

Cover Image, Volume 5, Issue 4

The cover image, by Lisa Maves and Jared Talbot, is based on the Focus Article Skeletal muscle fiber type: using insights from muscle developmental biology to dissect targets for susceptibility and resistance to muscle disease, DOI: 10.1002/wdev.230. image