Muscle fiber-type distribution predicts weight gain and unfavorable left ventricular geometry: a 19 year follow-up study

BACKGROUND: Skeletal muscle consists of type-I (slow-twitch) and type-II (fast-twitch) fibers, with proportions highly variable between individuals and mostly determined by genetic factors. Cross-sectional studies have associated low percentage of type-I fibers (type-I%) with many cardiovascular risk factors. METHODS: We investigated whether baseline type-I% predicts left ventricular (LV) structure and function at 19-year follow-up, and if so, which are the strongest mediating factors. At baseline in 1984 muscle fiber-type distribution (by actomyosin ATPase staining) was studied in 63 healthy men (aged 32–58 years). The follow-up in 2003 included echocardiography, measurement of obesity related variables, physical activity and blood pressure. RESULTS: In the 40 men not using cardiovascular drugs at follow-up, low type-I% predicted higher heart rate, blood pressure, and LV fractional shortening suggesting increased sympathetic tone. Low type-I% predicted smaller LV chamber diameters (P ≤ 0.009) and greater relative wall thickness (P = 0.034) without increase in LV mass (concentric remodeling). This was explained by the association of type-I% with obesity related variables. Type-I% was an independent predictor of follow-up body fat percentage, waist/hip ratio, weight gain in adulthood, and physical activity (in all P ≤ 0.001). After including these risk factors in the regression models, weight gain was the strongest predictor of LV geometry explaining 64% of the variation in LV end-diastolic diameter, 72% in end-systolic diameter, and 53% in relative wall thickness. CONCLUSION: Low type-I% predicts obesity and weight gain especially in the mid-abdomen, and consequently unfavourable LV geometry indicating increased cardiovascular risk

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle

<p>Abstract</p> <p>Background</p> <p>Skeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMD_J), a large animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMD_Jat various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.</p> <p>Results</p> <p>In comparison with TC muscles of CXMD_J, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMD_Jmuscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMD_Jdiaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMD_Jdiaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.</p> <p>Conclusion</p> <p>The CXMD_Jdiaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMD_Jdiaphragm was markedly different from that in <it>mdx </it>mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.</p

Expression of the myosin heavy chain IIB gene in porcine skeletal muscle: the role of the CArG-box promoter response element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and Ebox region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over- expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter

Divergence exists in the subcellular distribution of intramuscular triglyceride in human skeletal muscle dependent on the choice of lipid dye.

Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 μm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (- 24%) and central region (- 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (- 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis

EMG-Normalised Kinase Activation during Exercise Is Higher in Human Gastrocnemius Compared to Soleus Muscle

In mice, certain proteins show a highly confined expression in specific muscle groups. Also, resting and exercise/contraction-induced phosphorylation responses are higher in rat skeletal muscle with low mitochondrial content compared to muscles with high mitochondrial content, possibly related to differential reactive oxygen species (ROS)-scavenging ability or resting glycogen content. To evaluate these parameters in humans, biopsies from soleus, gastrocnemius and vastus lateralis muscles were taken before and after a 45 min inclined (15%) walking exercise bout at 69% VO2max aimed at simultaneously activating soleus and gastrocnemius in a comparable dynamic work-pattern. Hexokinase II and GLUT4 were 46–59% and 26–38% higher (p<0.05) in soleus compared to the two other muscles. The type I muscle fiber percentage was highest in soleus and lowest in vastus lateralis. No differences were found in protein expression of signalling proteins (AMPK subunits, eEF2, ERK1/2, TBC1D1 and 4), mitochondrial markers (F1 ATPase and COX1) or ROS-handling enzymes (SOD2 and catalase). Gastrocnemius was less active than soleus measured as EMG signal and glycogen use yet gastrocnemius displayed larger increases than soleus in phosphorylation of AMPK Thr172, eEF2 Thr56 and ERK 1/2 Thr202/Tyr204 when normalised to the mean relative EMG-signal. In conclusion, proteins with muscle-group restricted expression in mice do not show this pattern in human lower extremity muscle groups. Nonetheless the phosphorylation-response is greater for a number of kinase signalling pathways in human gastrocnemius than soleus at a given activation-intensity. This may be due to the combined subtle effects of a higher type I muscle fiber content and higher training status in soleus compared to gastrocnemius muscle

Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles

Characterization and Utilization of the Flexor Digitorum Brevis for Assessing Skeletal Muscle Function

Abstract Background The ability to assess skeletal muscle function and delineate regulatory mechanisms is essential to uncovering therapeutic approaches that preserve functional independence in a disease state. Skeletal muscle provides distinct experimental challenges due to inherent differences across muscle groups, including fiber type and size that may limit experimental approaches. The flexor digitorum brevis (FDB) possesses numerous properties that offer the investigator a high degree of experimental flexibility to address specific hypotheses. To date, surprisingly few studies have taken advantage of the FDB to investigate mechanisms regulating skeletal muscle function. The purpose of this study was to characterize and experimentally demonstrate the value of the FDB muscle for scientific investigations. Methods First, we characterized the FDB phenotype and provide reference comparisons to skeletal muscles commonly used in the field. We developed approaches allowing for experimental assessment of force production, in vitro and in vivo microscopy, and mitochondrial respiration to demonstrate the versatility of the FDB. As proof-of principle, we performed experiments to alter force production or mitochondrial respiration to validate the flexibility the FDB affords the investigator. Results The FDB is made up of small predominantly type IIa and IIx fibers that collectively produce less peak isometric force than the extensor digitorum longus (EDL) or soleus muscles, but demonstrates a greater fatigue resistance than the EDL. Unlike the other muscles, inherent properties of the FDB muscle make it amenable to multiple in vitro- and in vivo-based microscopy methods. Due to its anatomical location, the FDB can be used in cardiotoxin-induced muscle injury protocols and is amenable to electroporation of cDNA with a high degree of efficiency allowing for an effective means of genetic manipulation. Using a novel approach, we also demonstrate methods for assessing mitochondrial respiration in the FDB, which are comparable to the commonly used gastrocnemius muscle. As proof of principle, short-term overexpression of Pgc1α in the FDB increased mitochondrial respiration rates. Conclusion The results highlight the experimental flexibility afforded the investigator by using the FDB muscle to assess mechanisms that regulate skeletal muscle function

Myogenin Regulates Exercise Capacity and Skeletal Muscle Metabolism in the Adult Mouse

Although skeletal muscle metabolism is a well-studied physiological process, little is known about how it is regulated at the transcriptional level. The myogenic transcription factor myogenin is required for skeletal muscle development during embryonic and fetal life, but myogenin's role in adult skeletal muscle is unclear. We sought to determine myogenin's function in adult muscle metabolism. A Myog conditional allele and Cre-ER transgene were used to delete Myog in adult mice. Mice were analyzed for exercise capacity by involuntary treadmill running. To assess oxidative and glycolytic metabolism, we performed indirect calorimetry, monitored blood glucose and lactate levels, and performed histochemical analyses on muscle fibers. Surprisingly, we found that Myog-deleted mice performed significantly better than controls in high- and low-intensity treadmill running. This enhanced exercise capacity was due to more efficient oxidative metabolism during low- and high-intensity exercise and more efficient glycolytic metabolism during high-intensity exercise. Furthermore, Myog-deleted mice had an enhanced response to long-term voluntary exercise training on running wheels. We identified several candidate genes whose expression was altered in exercise-stressed muscle of mice lacking myogenin. The results suggest that myogenin plays a critical role as a high-level transcriptional regulator to control the energy balance between aerobic and anaerobic metabolism in adult skeletal muscle