Myosin Heavy Chain IIa Fibers Research Articles

Single Muscle Fiber Characteristics of The Oldest-Old

PURPOSE: To examine single muscle fiber function in the oldest-old. METHODS: Vastus lateralis muscle biopsies were obtained from five healthy independently living individuals (3F/2M, 89 ± 1 y) enrolled in the Health, Aging and Body Composition (Health ABC) Study. Isolated myosin heavy chain (MHC) I and IIa muscle fibers were examined for cross-sectional area (CSA) and contractile properties prior to MHC isoform identification via SDS-PAGE. RESULTS: MHC I muscle fibers were 21% larger (P < 0.05) than MHC IIa. A summary of muscle fiber characteristics is provided below (values are means ± SE). CONCLUSION: This investigation was the first to examine single muscle fiber contractile properties of independently living individuals > 85 y. In line with previous aging research, MHC IIa fibers were smaller than MHC I, indicative of MHC IIa-specific atrophy. Normalized power (an indicator of muscle fiber quality incorporating size, strength and speed) of MHC I fibers from these individuals, when compared to previous research from our lab using identical procedures, was 28% higher than healthy 25 y olds and similar to younger octogenarians (∼82 y). MHC IIa normalized power was 63% greater than 25 y olds and 39% greater than younger octogenarians. These data support the idea that power output per unit size (i.e. muscle quality) of surviving muscle fibers improves with advancing age, a phenomenon more pronounced in MHC IIa fibers. Thus, a likely key contributor to the loss of muscle function with age is a quantitative loss of whole muscle specific force rather than a reduction in muscle quality of remaining muscle fibers.Table: No title available.

Chronic disuse and skeletal muscle structure in older adults: sex-specific differences and relationships to contractile function.

In older adults, we examined the effect of chronic muscle disuse on skeletal muscle structure at the tissue, cellular, organellar, and molecular levels and its relationship to muscle function. Volunteers with advanced-stage knee osteoarthritis (OA, n = 16) were recruited to reflect the effects of chronic lower extremity muscle disuse and compared with recreationally active controls (n = 15) without knee OA but similar in age, sex, and health status. In the OA group, quadriceps muscle and single-fiber cross-sectional area were reduced, with the largest reduction in myosin heavy chain IIA fibers. Myosin heavy chain IIAX fibers were more prevalent in the OA group, and their atrophy was sex-specific: men showed a reduction in cross-sectional area, and women showed no differences. Myofibrillar ultrastructure, myonuclear content, and mitochondrial content and morphology generally did not differ between groups, with the exception of sex-specific adaptations in subsarcolemmal (SS) mitochondria, which were driven by lower values in OA women. SS mitochondrial content was also differently related to cellular and molecular functional parameters by sex: greater SS mitochondrial content was associated with improved contractility in women but reduced function in men. Collectively, these results demonstrate sex-specific structural phenotypes at the cellular and organellar levels with chronic disuse in older adults, with novel associations between energetic and contractile systems.

In Utero LPS Exposure Impairs Preterm Diaphragm Contractility

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Age-related slowing of myosin actin cross-bridge kinetics is sex specific and predicts decrements in whole skeletal muscle performance in humans

We hypothesize that age-related skeletal muscle dysfunction and physical disability may be partially explained by alterations in the function of the myosin molecule. To test this hypothesis, skeletal muscle function at the whole muscle, single fiber, and molecular levels was measured in young (21-35 yr) and older (65-75 yr) male and female volunteers with similar physical activity levels. After adjusting for muscle size, older adults had similar knee extensor isometric torque values compared with young, but had lower isokinetic power, most notably in women. At the single-fiber and molecular levels, aging was associated with increased isometric tension, slowed myosin actin cross-bridge kinetics (longer myosin attachment times and reduced rates of myosin force production), greater myofilament lattice stiffness, and reduced phosphorylation of the fast myosin regulatory light chain; however, the age effect was driven primarily by women (i.e., age-by-sex interaction effects). In myosin heavy chain IIA fibers, single-fiber isometric tension and molecular level mechanical and kinetic indexes were correlated with whole muscle isokinetic power output. Collectively, considering that contractile dysfunction scales up through various anatomical levels, our results suggest a potential sex-specific molecular mechanism, reduced cross-bridge kinetics, contributes to the reduced physical capacity with aging in women. Thus these results support our hypothesis that age-related alterations in the myosin molecule contribute to skeletal muscle dysfunction and physical disability and indicate that this effect is stronger in women.

Concurrent aerobic exercise interferes with the satellite cell response to acute resistance exercise

The addition of aerobic exercise (AE) to a resistance exercise (RE) program (concurrent exercise, CE) can interfere with maximum muscle fiber growth achieved with RE. Further, CE appears to markedly affect the growth of myosin heavy chain (MHC) I, but not MHC IIa fibers. The mechanism responsible for this "interference" is unclear. Satellite cell (SC) responsiveness to exercise appears to influence muscle adaptation but has not yet been examined following acute concurrent exercise. Thus, we assessed the fiber-type-specific SC response to RE, AE, and CE exercise. Eight college-aged males completed the following two exercise trials: the RE trial, which consisted of unilateral leg extensions and presses (4 sets ≥ 10 repetitions: 75% 1 repetition maximum, RM); and the AE/CE trial, which included an identical RE protocol with the opposite leg, immediately followed by subjects cycling for 90 min (60% W(max)). Muscle biopsies were obtained from the vastus lateralis before and 4 days after each session. Samples were cross-sectioned, stained with antibodies against NCAM, Ki-67, and MHC I, counterstained with DAPI, and analyzed for SC density (SC per fiber), SC activation, and fiber type. SC density increased to a greater extent following RE (38 ± 10%), compared with CE (-6 ± 8%). Similarly, MHC I muscle fiber SC density displayed a greater increase following RE (46 ± 14%), compared with AE (-7 ± 17%) and CE (-8 ± 8%). Our data indicate that the SC response to RE is blunted when immediately followed by AE, at least in MHC I muscle fibers, and possibly MHC II fibers. This suggests that the physiological environment evoked by AE might attenuate the eventual addition of myonuclei important for maximum muscle fiber growth and consequent force-producing capacity.

Myosin Heavy Chain Plasticity in Aging Skeletal Muscle with Aerobic Exercise Training

PURPOSE: To comprehensively assess myosin heavy chain (MHC) plasticity in aging skeletal muscle in response to aerobic exercise training. METHODS: Muscle biopsy samples were obtained from the vastus lateralis of eight older women (70±2y) before and after 12-wks of cycle ergometer training. MHC composition was measured at the mRNA level via qPCR and at the protein level in muscle homogenates and individual myofibers using SDS-PAGE. RESULTS: At the mRNA level, aerobic training reduced (p<0.05) MHC IIx and tended to elevate (p=0.10) MHC I mRNA expression. The homogenate proportion of MHC I protein increased (54±4 to 61±2%, p<0.05) and MHC IIx protein decreased (8±1 to 4±1%, p<0.05) after training while the MHC IIa proportion was unaltered. Single myofiber analysis demonstrated a robust elevation (42±4 to 52±3%, p<0.05) in the percentage of MHC I fibers with a reciprocal decrease (48±3 to 38±4% p<0.05) in the percentage of MHC IIa fibers after training. CONCLUSION: These results document a reduction in the relative amount of fast MHC isoforms (MHC IIa or IIx) with a concomitant increase in slow MHC I isoforms independent of the method employed. The shift to an oxidative myosin heavy chain phenotype indicates that aerobic exercise training augments skeletal muscle health of older women and may serve as a countermeasure to age-related skeletal muscle atrophy. Supported by NIH AG032127

A new method to study in vivo protein synthesis in slow- and fast-twitch muscle fibers and initial measurements in humans

The aim of this study was to develop an approach to directly assess protein fractional synthesis rate (FSR) in isolated human muscle fibers in a fiber type-specific fashion. Individual muscle fibers were isolated from biopsies of the vastus lateralis (VL) and soleus (SOL) obtained from eight young men during a primed, continuous infusion of [5,5,5-(2)H3]leucine performed under basal conditions. To determine mixed protein FSR, a portion of each fiber was used to identify fiber type, fibers of the same type were pooled, and the [5,5,5-(2)H3]leucine enrichment was determined via GC-MS. Processing isolated slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) fibers for mixed protein bound [5,5,5-(2)H3]leucine enrichment yielded mass ion chromatographic peaks that were similar in shape, abundance, and measurement reliability as tissue homogenates. In the VL, MHC I fibers exhibited a 33% faster (P<0.05) mixed protein FSR compared with MHC IIa fibers (0.068+/-0.006 vs. 0.051+/-0.003%/h). MHC I fibers from the SOL (0.060+/-0.005%/h) and MHC I fibers from the VL displayed similar (P>0.05) mixed protein FSR. Feasibility of processing isolated human muscle fibers for analysis of myofibrillar protein [5,5,5-(2)H3]leucine enrichment was also confirmed in non-fiber-typed pooled fibers from the VL. These methods can be applied to the study of fiber type-specific responses in human skeletal muscle. The need for this level of investigation is underscored by the different contributions of each fiber type to whole muscle function and the numerous distinct adaptive functional and metabolic changes in MHC I and MHC II fibers originating from the same muscle.

Mechanisms Underlying Skeletal Muscle Weakness in Human Heart Failure

Background— Patients with chronic heart failure (HF) frequently experience skeletal muscle weakness that limits physical function. The mechanisms underlying muscle weakness, however, have not been clearly defined. Methods and Results— This study examined the hypothesis that HF promotes a loss of myosin protein from single skeletal muscle fibers, which in turn reduces contractile performance. Ten patients with chronic HF and 10 controls were studied. Muscle atrophy was not evident in patients, and groups displayed similar physical activity levels, suggesting that observed differences reflect the effects of HF and not muscle atrophy or disuse. In single muscle fibers, patients with HF showed reduced myosin heavy chain protein content ( P &lt;0.05) that manifested as a reduction in functional myosin-actin cross-bridges ( P &lt;0.05). No evidence was found for a generalized loss of myofilament protein, suggesting a selective loss of myosin. Accordingly, single muscle fiber maximal Ca 2+ -activated tension was reduced in myosin heavy chain I fibers in patients ( P &lt;0.05). However, tension was maintained in myosin heavy chain IIA fibers in patients because a greater proportion of available myosin heads were bound to actin during Ca 2+ activation ( P &lt;0.01). Conclusions— Collectively, our results show that HF alters the quantity and functionality of the myosin molecule in skeletal muscle, leading to reduced tension in myosin heavy chain I fibers. Loss of single fiber myosin protein content represents a potential molecular mechanism underlying muscle weakness and exercise limitation in patients with HF.

Single muscle fiber contractile properties of young competitive distance runners

The purpose of this investigation was to characterize the contractile properties of individual slow- and fast-twitch myofibers from highly trained distance runners. Muscle biopsies were obtained from the gastrocnemius of eight competitive runners (Run) and eight recreationally active individuals (Rec). Slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) myofibers were isolated and analyzed for diameter (microm), peak force (Po; mN), unloaded contraction velocity (Vo; fiber lengths/s), and power. Maximum oxygen uptake was higher (P<0.05) in Run (71+/-1 vs. 47+/-2 ml.kg(-1).min(-1)). Diameter of MHC I and MHC IIa fibers from Run subjects was approximately 20% greater (P<0.05) than Rec. Peak force of the MHC IIa fibers was 31% higher (P<0.05) in Run, whereas Po of MHC I fibers was not different between groups. No differences for specific tension (Po/cross-sectional area) were present between groups for either fiber type. Vo was higher (P<0.05) in MHC I (+70%) and MHC IIa (+18%) fibers from Run subjects. In vitro peak absolute power (microN.s(-1)) of both fiber types was greater (P<0.05) in Run (131 and 85% for MHC I and MHC IIa, respectively). Additionally, normalized power (W/l) of the MHC I fibers was 64% higher in Run, whereas no differences were noted for normalized power of MHC IIa fibers. These data indicate that highly trained endurance runners have elevated contraction velocity in both slow- and fast-twitch myofibers. These characteristics of the fast-twitch muscle fibers have not been previously reported in competitive endurance athletes and may contribute to the high level of running performance in these athletes.

Single muscle fiber gene expression with resistance training in young women

The purpose of this investigation was to examine fiber type specific gene expression (GE) before and after 12 weeks (36 sessions) of progressive resistance training (PRT) for the knee extensors. Muscle biopsies were taken from vastus lateralis of 8 young women (23 ± 2 y, 67 ± 5 kg) before and 4 h post the 1st(acute) and 36th (chronic) training session. Myogenic (MyoD, myogenin, MRF4) and proteolytic (atrogin‐1, MuRF‐1, myostatin) GE was measured in mRNA from pooled myosin heavy chain (MHC) I and IIa fibers, using real‐time RT PCR. After the 1st training session, myogenin and MRF4 GE induction (p&lt;0.05) was targeted to the MHC I fibers, while myostatin GE decreased (p&lt;0.05) and MuRF‐1 GE increased (p&lt;0.05) in both fiber types. After 12 weeks of training the basal level GE of myogenin (MHC I) and MRF4 (MHC I and IIa) increased (p&lt;0.05). In response to the 36th training bout, myogenin GE induction (p&lt;0.05) and myostatin GE reduction (p&lt;0.05) was targeted to the MHC IIa fibers. There was a trend (p=0.07) for MuRF‐1 to increase in both fiber types after the 36th session. In summary, these data suggest that 12 weeks of PRT alters basal level myogenic GE. Interestingly, the MHC IIa specific response for myogenin and myostatin in the chronic phase may be important for the fast‐twitch fiber adaptation that occurs with resistance training in young individuals. Supported by NIH grant AG18409.

Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers

The purpose of this study was to characterize changes in mRNA expression of select proteolytic markers in human slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) single skeletal muscle fibers following a bout of resistance exercise (RE). Muscle biopsies were obtained from the vastus lateralis of eight young healthy sedentary men [23 +/- 2 yr (mean +/- SD), 93 +/- 17 kg, 183 +/- 6 cm] before and 4 and 24 h after 3 x 10 repetitions of bilateral knee extensions at 65% of one repetition maximum. The mRNA levels of TNF-alpha, calpains 1 and 2, muscle RING (really interesting novel gene) finger-1 (MuRF-1), atrogin-1, caspase-3, B-cell leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) were quantified using real-time RT-PCR. Generally, MHC I fibers had higher (1.6- to 5.0-fold, P < 0.05) mRNA expression pre- and post-RE. One exception was a higher (1.6- to 3.9-fold, P < 0.05) Bax-to-Bcl-2 mRNA ratio in MHC IIa fibers pre- and post-RE. RE increased (1.4- to 4.8-fold, P < 0.05) MuRF-1 and caspase-3 mRNA levels 4-24 h post-RE in both fiber types, whereas Bax-to-Bcl-2 mRNA ratio increased 2.2-fold (P < 0.05) at 4 h post-RE only in MHC I fibers. These results suggest that MHC I fibers have a greater proteolytic mRNA expression pre- and post-RE compared with MHC IIa fibers. The greatest mRNA induction following RE was in MuRF-1 and caspase-3 in both fiber types. This altered and specific proteolytic mRNA expression among slow- and fast-twitch muscle fibers indicates that the ubiquitin/proteasomal and caspase pathways may play an important role in muscle remodeling with RE.

Upper body training and the triceps brachii muscle of elite cross country skiers

This study aimed at evaluating whether addition of extensive upper body training in well-trained cross country skiers induces an adaptation of the triceps brachii (TB) muscle and whether this affects performance. Muscle biopsies were obtained from TB muscle in six male elite cross country skiers before and after 20 weeks of increased upper body training. The cross-sectional area of type I and IIA fibers increased by 11.3% and 24.0%, respectively, and so did the number of capillaries per fiber (2.3-3.2) (all P<0.05). SDS-polyacrylamide electrophoresis revealed in single fibers that the number of fibers expressing myosin heavy chain (MHC) type I isoform decreased from 68.7% to 60.9% (P<0.05), MHC I/IIA isoform was unaltered, while MHC IIA fibers increased from 21.6% to 35.7% and the 4.8% MHC IIA/IIX disappeared with the training (both P<0.05). Citrate synthase and 3-hydroxyacyl coenzyme A dehydrogenase activities increased by 23.3% and 15.4%, respectively, and double poling 10 km time-trial by 10.4% (all P<0.05). The values for TB are similar to what has been demonstrated for leg muscles after exercise training. The subjects who demonstrated the largest improvement in performance exhibited the largest muscle adaptation, which, in turn, was related to the pre-maximal oxygen uptake.

Alterations in single muscle fiber calcium sensitivity with resistance training in older women.

The purpose of this investigation was to determine the effects of a 12-week progressive resistance-training program (PRT) on single muscle fiber calcium sensitivity in six older women (73 +/- 2 years). Muscle biopsy samples of the vastus lateralis were obtained pre- and post-PRT. Chemically skinned single muscle fibers ( n=274) were dissected and studied. The experimental sequence for each fiber was the determination of peak maximal isometric tension ( P(o)) at pCa 4.5 (pCa=-log[Ca(2+)]), and then subsequent submaximal activations of the fiber at nine Ca(2+) concentrations (pCa 6.8 to 4.7). Myosin heavy chain (MHC) I fiber (slow-twitch) diameter increased 16% ( P<0.05) with no change in MHC IIa fibers (fast-twitch) pre- to post-PRT, respectively. P(o) in MHC I fibers increased 34% ( P<0.05) as a result of the training with no change in MHC IIa fibers. The mean MHC I Ca(2+) activation threshold (minimal amount of Ca(2+) necessary to induce tension) increased from 6.83 +/- 0.02 to 6.91 +/- 0.01 ( P<0.05), as did the mean half-maximal activation (pCa(50)), 5.51 +/- 0.02 to 5.71 +/- 0.03 ( P<0.05) with PRT. The slope of the Hill plot above ( n(1)) the pCa(50) for MHC I did not change significantly with the PRT. However, the slope of the Hill plot below ( n(2)) the pCa(50) for MHC I demonstrated an increase ( P<0.05) with training. There were no differences with MHC IIa fibers with PRT for any of the variables measured. In conclusion, the results of this investigation indicate that myofibril Ca(2+) sensitivity and activation properties are altered in MHC I, but not MHC IIa fibers with PRT in older women. The alterations in the MHC I Ca(2+) properties appear to have an effect on the mechanisms involved with skeletal muscle adaptability in older women following PRT.

Effect of resistance training on single muscle fiber contractile function in older men.

The purpose of this study was to examine single cell contractile mechanics of skeletal muscle before and after 12 wk of progressive resistance training (PRT) in older men (n = 7; age = 74 +/- 2 yr and weight = 75 +/- 5 kg). Knee extensor PRT was performed 3 days/wk at 80% of one-repetition maximum. Muscle biopsy samples were obtained from the vastus lateralis before and after PRT (pre- and post-PRT, respectively). For analysis, chemically skinned single muscle fibers were studied at 15 degrees C for peak tension [the maximal isometric force (P(o))], unloaded shortening velocity (V(o)), and force-velocity parameters. In this study, a total of 199 (89 pre- and 110 post-PRT) myosin heavy chain (MHC) I and 99 (55 pre- and 44 post-PRT) MHC IIa fibers were reported. Because of the minimal number of hybrid fibers identified post-PRT, direct comparisons were limited to MHC I and IIa fibers. Muscle fiber diameter increased 20% (83 +/- 1 to 100 +/- 1 microm) and 13% (86 +/- 1 to 97 +/- 2 microm) in MHC I and IIa fibers, respectively (P < 0.05). P(o) was higher (P < 0.05) in MHC I (0.58 +/- 0.02 to 0.90 +/- 0.02 mN) and IIa (0.68 +/- 0.02 to 0.85 +/- 0.03 mN) fibers. Muscle fiber V(o) was elevated 75% (MHC I) and 45% (MHC IIa) after PRT (P < 0.05). MHC I and IIa fiber power increased (P < 0.05) from 7.7 +/- 0.5 to 17.6 +/- 0.9 microN. fiber lengths. s(-1) and from 25.5 to 41.1 microN. fiber lengths. s(-1), respectively. These data indicate that PRT in elderly men increases muscle cell size, strength, contractile velocity, and power in both slow- and fast-twitch muscle fibers. However, it appears that these changes are more pronounced in the MHC I muscle fibers.