Myosin Heavy Chain Isoforms Research Articles

Evaluation of Guanidinoacetic Acid Supplementation on Finishing Beef Steer Growth Performance, Skeletal Muscle Cellular Response, and Carcass Characteristics.

This study elucidated the effects of dosage-dependent guanidinoacetic acid (GAA) supplementation on growth performance, muscle responses, and carcass characteristics in finishing beef steers. Thirty crossbred Red Angus beef steers (395 ± 28.09kg) were randomly assigned one of three treatments during a 146-day feedlot study: basal diet without GAA supplementation (CONTROL), 1g of GAA per 100kg of BW daily (LOWGAA), and 2g of GAA per 100kg of BW daily (HIGHGAA). Individual feed intake was monitored daily, growth performance parameters were collected every 28 days, and longissimus muscle (LM) biopsies occurred every 56 days. In biopsied LM, greater (P = 0.048) mRNA expression of IGF-1 was observed in LOWGAA steers on d 112 compared to the CONTROL group. LOWGAA steers also exhibited greater expression of myosin heavy chain (MHC) I compared to CONTROL steers (P < 0.05) and MHC IIA compared to both CONTROL and HIGHGAA treatment groups (P < 0.01) on d 112. GAA supplementation resulted in no change in carcass characteristics, serum and LM tissue metabolites, LM composition, and Warner-Bratzler shear force (WBSF) values (P > 0.05). Data collected from this study demonstrate the influence of GAA supplementation on the gene expression of MHC isoforms and their role in skeletal muscle growth, differentiation, and muscle fiber-typing.

Exercise, disease state and sex influence the beneficial effects of Fn14-depletion on survival and muscle pathology in the SOD1G93A amyotrophic lateral sclerosis (ALS) mouse model

BackgroundAmyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1G93A ALS mice could lead to differential and potentially improved benefits.MethodsWe thus investigated the contribution of Fn14 to disease phenotypes in the SOD1G93A ALS mice. To do so, Fn14 knockout mice (Fn14−/−) were crossed onto the SOD1G93A background to generate SOD1G93A;Fn14−/− mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14−/−, SOD1G93A and SOD1G93A;Fn14−/−).ResultsHere, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1G93A mice. We then show that Fn14-depleted SOD1G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size.ConclusionsOur study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies.

Effects of age on human skeletal muscle: A systematic review and meta-analysis of myosin heavy chain isoform protein expression, fiber size and distribution.

Human studies examining the cellular mechanisms behind sarcopenia, or age-related loss of skeletal muscle mass and function, have produced inconsistent results. A systematic review and meta-analysis were performed to determine the aging effects on protein expression, size and distribution of fibers with various myosin heavy chain (MyHC) isoforms. Study eligibility included MyHC comparisons between young (18-49 years) and older (≥ 60 years) adults, with 27 studies identified. Relative protein expression was higher with age for the slow-contracting MyHC I fibers, with correspondingly lower fast-contracting MyHC II and IIA values. Fiber sizes were similar with age for MyHC I, while smaller for MyHC II and IIA. Fiber distributions were similar with age. When separated by sex, the few studies that examined females showed atrophy of MyHC II and IIA fibers with age, but no change in MyHC protein expression. Additional analyses by measurement technique, physical activity, and muscle biopsied provided important insights. In summary, age-related atrophy in fast-contracting fibers lead to more of the slow-contracting, lower force-producing isoform in older male muscles, which helps explain their age-related loss in whole muscle force, velocity, and power. Exercise or pharmacological interventions that shift MyHC expression towards faster isoforms and/or increase fast-contracting fiber size should decrease the prevalence of sarcopenia. Our findings also indicate that future studies need to include or focus solely on females, measure MyHC IIA and IIX isoforms separately, examine fiber type distribution, sample additional muscles to the vastus lateralis, and incorporate an objective measurement of physical activity.

Skeletal muscle myosin heavy chain fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n=8; 5±2years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24h after their first/naïve RE bout. Participants also engaged in 10weeks of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined in an acute cycling study (n=7) and a study involving 2weeks of leg immobilization (n=20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3h post-RE (∼200%, P=0.018) and returned to pre-exercise levels by 6h post-RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3h post-RE (8.6±6.3-fold vs. 2.1±0.7-fold, P=0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, P<0.001); however, the last RE bout response was attenuated compared to the first bout (P=0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P<0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease-associated muscle atrophy affect these outcomes is needed. HIGHLIGHTS: What is the central question of this study? How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation. What is the main finding and its importance? This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases.

Effect of the combination of Lithospermum erythrorhizon and Lonicera japonica on dexamethasone-induced muscle atrophy in mice

AbstractSkeletal muscle atrophy occurs in several pathological conditions. Among other reasons, high-dose or long-term administration of glucocorticoids increases circulating glucocorticoid levels and causes muscle atrophy. The purpose of this study was to investigate whether Lithospermum erythrorhizon and Lonicera japonica complex extract (LELJ) has a beneficial effect on dexamethasone (Dexa)-induced muscle atrophy. In Dexa-induced myotube atrophy, treatment with LELJ increased myotube diameter, decreased the expression of muscle atrophy markers, and increased the expression of myosin heavy chain (MHC) isoforms. Supplementation with LELJ improved muscle function and performance in mice with Dexa-induced muscle atrophy as demonstrated by grip strength and running tests. Additionally, it increased skeletal muscle mass, size, and expression of MHC isoforms and protein synthesis-related markers. Furthermore, it reduced the upregulated protein levels of skeletal muscle atrophy markers in Dexa-treated mice. Supplementation with LELJ reversed Dexa-induced translocation of the glucocorticoid receptor and forkhead box O3 from the cytosol to the nucleus in skeletal muscles. LELJ also ameliorated age-related muscle loss by extending lifespan and increasing locomotor capacity in Caenorhabditis elegans. We identified loganin and lithospermic acid as bioactive compounds of LELJ and found that treatment with these agents increased myotube diameter, MHC isoform, and puromycin protein levels, and decreased atrophy markers in Dexa-treated myotubes. The current findings underscore how LELJ can prevent Dexa-induced skeletal muscle atrophy, attributing the effects to loganin and lithospermic acid.

Endurance exercise-induced histone methylation modification involved in skeletal muscle fiber type transition and mitochondrial biogenesis

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.

Fiber-type composition and 3D capillary analysis of the human splenius capitis muscle.

Despite the significance of neck muscles in musculoskeletal disorders, their microscopic anatomy remains poorly characterized. This study examined the splenius capitis muscle, focusing on its fiber-type composition, fiber size, and capillary network characteristics. For comparison and validation, the vastus lateralis muscle was also analyzed. Muscle samples from 13 young male subjects (mean age ± SD: 35.7 ± 8.6 years) were collected within 24-h post-mortem during autopsy. Myosin heavy chain (MyHC) isoform expression was characterized immunohistochemically in 10 μm sections, while the capillary network architecture was assessed in 100 μm sections. Immunofluorescence staining, confocal microscopy, and 3D image analysis were employed to quantify capillary tortuosity, anisotropy, branch density (Br dens), and the length of capillaries per muscle volume (LV), per muscle fiber length (LL), per fiber surface area (LS), and per fiber volume (LVf). Compared to the vastus lateralis muscle, the splenius capitis muscle had a higher percentage of type 1 fibers (51.2% vs 39.7%), fewer type 2a fibers (16.2% vs 31.4%), and smaller fiber diameters (35.5-40.9 μm vs 47-56.1 μm). It also displayed lower Br dens (P = 0.0069), higher anisotropy (P = 0.0004), and lower LL (P < 0.0001) but higher LVf (P = 0.0486). In the splenius capitis muscle, body mass index (BMI) negatively correlated with LV (P = 0.0155), LS (P = 0.0091), LVf (P = 0.0137), and anisotropy (P = 0.0425), and positively correlated with tortuosity (P = 0.0473), indicating a reduction in the capillary network. In the vastus lateralis muscle, only LV (P = 0.0161) decreased with high BMI. This study characterized the fiber-type composition, fiber size, and 3D capillary network of the splenius capitis muscle, establishing a baseline for investigations into pathological muscle alterations.

Effects of Short-Term Treatment of Rabbit Extraocular Muscle With Ciliary Neurotrophic Factor.

Little is known about the effect of ciliary neurotrophic factor (CNTF) on extraocular muscles, but microarray studies suggested CNTF might play a role in the development and/or maintenance of strabismus. The effect of short-term treatment of adult rabbit extraocular muscle with injected CNTF was examined for its ability to alter muscle characteristics. Eight adult New Zealand white rabbits received an injection into one superior rectus muscle of 2 µg/100 µL CNTF on 3 consecutive days. One week after the first injection, the rabbits were euthanized, and the treated and contralateral superior rectus muscles were assessed for force generation capacity and contraction characteristics using an in vitro stimulation protocol and compared to naïve control superior rectus muscles. All muscles were analyzed to determine mean cross-sectional areas and expression of slow twitch myosin heavy chain isoform. Short-term treatment of rabbit superior rectus muscles with CNTF resulted in a significant decrease in muscle force generation, but only at the higher stimulation frequencies. Significantly decreased myofiber cross-sectional areas of the treated muscles correlated with the decreased generated force. In addition, there were significant changes to contractile properties of the treated muscles, as well as a decrease in the number of myofibers expressing slow twitch myosin heavy chain. We show that short-term treatment of a single rabbit superior rectus muscle results in decreased myofiber size, decreased force, and altered contractile characteristics. Further studies are needed to determine if it can play a role in improving alignment in animal models of strabismus.

Abnormal expression of myosin heavy chains in early postnatal stages of spinal muscular atrophy type I at single fibre level.

We investigated myosin heavy chain (MyHC) isoform expression at early postnatal stages of clinically and genetically confirmed spinal muscular atrophy type 1 (SMA1) patients, in order to study the muscle fibre differentiation compared to age-matched controls at single fibre level. Open skeletal muscle biopsies were performed from the quadriceps muscle in four SMA1 patients and three age-matched controls. Standard techniques were used for immunohistochemistry of embryonic and foetal MyHCs. Type I, IIa and IIx MyHCs were assessed by applying quadruple immunofluorescence. Western blot was performed to analyse the amount of survival motor neuron (SMN) protein in the muscle samples. There were profound and early alterations in MyHC expression from 7 days of life compared to age-matched controls. The expression of type IIx MyHC was completely lost in SMA1 and instead developmental isoforms remained highly expressed. Foetal MyHC was still, at 3.5 months of age, expressed in the majority of muscle fibres in SMA1 patients, whereas it was completely downregulated in age-matched controls. The level of SMN protein was reduced in all SMN1 patients. The abnormal pattern of MyHC expression in postnatal stages of SMA1 was observed early in the newborn period, which may have implications for the effects of gene therapy, since there are clear clinical benefits from early treatment. Whether such aberrant and delayed expression of MyHCs can be completely restored by postnatal gene therapy remains to be studied and may also have implications for new phenotypes that will evolve with new therapies.

Can non-invasive motor unit analysis reveal distinct neural strategies of force production in young with uncomplicated type 1 diabetes?

to investigate the early consequences of type 1 diabetes (T1D) on the neural strategies of muscle force production. motor unit (MU) activity was recorded from the vastus lateralis muscle with High-Density surface Electromyography during isometric knee extension at 20 and 40% of maximum voluntary contraction (MVC) in 8 T1D (4 males, 4 females, 30.5 ± 3.6years) and 8 matched control (4 males, 4 females, 27.3 ± 5.9years) participants. Muscle biopsies were also collected from vastus lateralis for fiber type analysis, including myosin heavy chain (MyHC) isoform content via protein and mRNA expression. MVC was comparable between groups as well as MU conduction velocity, action potentials' amplitude and proportions of MyHC protein isoforms. Nonetheless, MU discharge rate, relative derecruitment thresholds and mRNA expression of MyHC isoform I were lower in T1D. young people with uncomplicated T1D present a different neural control of muscle force production. Furthermore, differences are detectable non-invasively in absence of any functional manifestation (i.e., force production and fiber type distribution). These novel findings suggest that T1D has early consequences on the neuromuscular system and highlights the necessity of a better characterization of neural control in this population.

Abstract Mo118: Myofilament proteolysis may underlie contractile remodeling in atrial fibrillation

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function, including decreased blood flow velocity and reduced atrial contractility, known as atrial stunning, occurs. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. In atrial cardiomyocytes that were useable for skinned single cardiomyocyte force-calcium studies, we found reduced force of contraction. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, MHC, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed increased calcium sensitivity that was consistent with increased cTnI degradation and decreased TnI phosphorylation. These results provide an understanding of the contractile remodeling that occurs in AFib.

Cardiac Atrophy, Dysfunction, and Metabolic Impairments: A Cancer-Induced Cardiomyopathy Phenotype

Muscle atrophy and weakness are prevalent features of cancer. Although extensive research has characterized skeletal muscle wasting in cancer cachexia, limited studies have investigated how cardiac structure and function are affected by therapy-naive cancer. Herein, orthotopic, syngeneic models of epithelial ovarian cancer and pancreatic ductal adenocarcinoma, and a patient-derived pancreatic xenograft model, were used to define the impact of malignancy on cardiac structure, function, and metabolism. Tumor-bearing mice developed cardiac atrophy and intrinsic systolic and diastolic dysfunction, with arterial hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased, and carbohydrate-supported respiration increased-showcasing a substrate shift in cardiac metabolism that is characteristic of heart failure. Epithelial ovarian cancer decreased cytoskeletal and cardioprotective gene expression, which was paralleled by down-regulation of transcription factors that regulate cardiomyocyte size and function. Patient-derived pancreatic xenograft tumor-bearing mice show altered myosin heavy chain isoform expression-also a molecular phenotype of heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated by cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, two cancer types were used to cross-validate evidence of the structural, functional, and metabolic cancer-induced cardiomyopathy, thus providing translational evidence that could impact future medical management strategies for improved cancer recovery in patients.

Does sex matter in the cheetah? Insights into the skeletal muscle of the fastest land animal.

The cheetah is considered the fastest land animal, but studies on their skeletal muscle properties are scarce. Vastus lateralis biopsies, obtained from male and female cheetahs as well as humans, were analysed and compared for fibre type and size, and metabolism. Overall, cheetah muscle had predominantly type IIX fibres, which was confirmed by the myosin heavy chain isoform content (mean±s.d. type I: 17±8%, type IIA: 21±6%, type IIX: 62±12%), whereas human muscle contained predominantly type I and IIA fibres (type I: 49±14%, type IIA: 43±8%, type IIX: 7±7%). Cheetahs had smaller fibres than humans, with larger fibres in the males compared with their female counterparts. Citrate synthase (16±6 versus 28±7 µmolmin-1g-1 protein, P<0.05) and 3-hydroxyacyl co-enzyme A dehydrogenase (30±11 versus 47±15 µmolmin-1g-1 protein, P<0.05) activities were lower in cheetahs than in humans, whereas lactate dehydrogenase activity was 6 times higher in cheetahs (2159±827 versus 382±161 µmolmin-1g-1 protein, P<0.001). The activities of creatine kinase (4765±1828 versus 6485±1298, P<0.05 µmolmin-1g-1 protein) and phosphorylase (111±29 versus 216±92 µmolmin-1g-1 protein) were higher in humans, irrespective of the higher type IIX fibres in cheetahs. Superoxide dismutase and catalase, markers of antioxidant capacity, were higher in humans, but overall antioxidant capacity was higher in cheetahs. To conclude, fibre type, fibre size and metabolism differ between cheetahs and humans, with limited differences between the sexes.

Exploring the Longissimus Muscle: Unraveling its Correlation with Meat Quality in Bos indicus and Crossbred Bulls.

This study investigated muscle tissue in Bos indicus and crossbred bulls to explain differences in meat quality traits. Carcass traits, meat quality parameters, and biochemical and molecular investigations of myofibrillar proteins are described. Methods for evaluating pH, intramuscular fat (IMF), meat color (L*, a*, b*), water losses, tenderness, and molecular biology assays have been outlined. Specific procedures detailing calibration, sample preparation, and data analysis for each method are described. These include techniques such as infrared spectroscopy for IMF content, objective tenderness assessment, and electrophoretic separation of MyHC isoforms. Color parameters were highlighted as potential tools for predicting beef tenderness, a crucial quality trait influencing consumer decisions. The study employed the Warner-Bratzler shear force (WBSF) method, revealing values of 4.68 and 4.23 kg for Nellore and Angus-Nellore (P < 0.01), respectively. Total cooking losses and biochemical analyses, including myofibril fragmentation index (MFI), provided insights into tenderness variations. Muscle fiber types, particularly myosin heavy chain (MyHC) isoforms, were investigated, with a notable absence of MyHC-IIb isoform in the studied Zebu animals. The relationship between MyHC-I and meat tenderness revealed divergent findings in the literature, highlighting the complexity of this association. Overall, the study provides comprehensive insights into the factors influencing meat quality in Bos indicus and crossbred (Bos taurus × Bos indicus) bulls, offering valuable information for the beef industry.

Sucrose-induced metabolic syndrome differentially affects energy metabolism and fiber phenotype of EDL and soleus muscles during exercise in the rat.

Molecular mechanisms associated to improvement of metabolic syndrome (MetS) during exercise are not fully elucidated. MetS was induced in 250 g male Wistar rats by 30% sucrose in drinking water. Control rats receiving tap water were controls, both groups received solid standard diet. After 14 weeks, an endurance exercised group, and a sedentary were formed for 8 weeks. The soleus and extensor digitorum longus (EDL) muscles were dissected to determine contractile performance, expression of myosin heavy chain isoforms, PGC1α, AMPKα2, NFATC1, MEF2a, SIX1, EYA1, FOXO1, key metabolic enzymes activities. Exercise mildly improved MetS features. MetS didn't alter the contractile performance of the muscles. Exercise didn't altered expression of PGC1α, NFATC1, SIX1 and EYA1 on MetS EDL whereas NFATC1 increased in soleus. Only citrate synthase was affected by MetS on the EDL and this was partially reverted by exercise. Soleus α-ketoglutarate dehydrogenase activity was increased by exercise but MetS rendered the muscle resistant to this effect. MetS affects mostly the EDL muscle, and endurance exercise only partially reverts this. Soleus muscle seems more resilient to MetS. We highlight the importance of studying both muscles during MetS, and their metabolic remodeling on the development and treatment of MetS by exercise.

High-intensity interval training using electrical stimulation ameliorates muscle fatigue in chronic kidney disease-related cachexia by restoring mitochondrial respiratory dysfunction.

Exercise, especially high-intensity interval training (HIIT), can increase mitochondrial respiratory capacity and enhance muscular endurance, but its systemic burden makes it difficult to safely and continuously prescribe for patients with chronic kidney disease (CKD)-related cachexia who are in poor general condition. In this study, we examined whether HIIT using electrical stimulation (ES), which does not require whole-body exercise, improves muscle endurance in the skeletal muscle of 5/6 nephrectomized rats, a widely used animal model for CKD-related cachexia. Male Wistar rats (10weeks old) were randomly assigned to a group of sham-operated (Sham) rats and a group of 5/6 nephrectomy (Nx) rats. HIIT was performed on plantar flexor muscles in vivo with supramaximal ES every other day for 4weeks to assess muscle endurance, myosin heavy-chain isoforms, and mitochondrial respiratory function in Nx rats. A single session was also performed to identify upstream signaling pathways altered by HIIT using ES. In the non-trained plantar flexor muscles from Nx rats, the muscle endurance was significantly lower than that in plantar flexor muscles from Sham rats. The proportion of myosin heavy chain IIa/x, mitochondrial content, mitochondrial respiratory capacity, and formation of mitochondrial respiratory supercomplexes in the plantaris muscle were also significantly decreased in the non-trained plantar flexor muscles from Nx rats than compared to those in plantar flexor muscles from Sham rats. Treatment with HIIT using ES for Nx rats significantly improved these molecular and functional changes to the same degrees as those in Sham rats. Furthermore, a single session of HIIT with ES significantly increased the phosphorylation levels of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK), pathways that are essential for mitochondrial activation signaling by exercise, in the plantar muscles of both Nx and Sham rats. The findings suggest that HIIT using ES ameliorates muscle fatigue in Nx rats via restoration of mitochondrial respiratory dysfunction with activation of AMPK and p38 MAPK signaling. Our ES-based HIIT protocol can be performed without placing a burden on the whole body and be a promising intervention that is implemented even in conditions of reduced general performance status such as CKD-related cachexia.

Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Extreme Tolerance of Extraocular Muscles to Diseases and Aging: Why and How?

The extraocular muscles (EOMs) possess unique characteristics that set them apart from other skeletal muscles. These muscles, responsible for eye movements, exhibit remarkable resistance to various muscular dystrophies and aging, presenting a significant contrast to the vulnerability of skeletal muscles to these conditions. In this review, we delve into the cellular and molecular underpinnings of the distinct properties of EOMs. We explore their structural complexity, highlighting differences in fiber types, innervation patterns, and developmental origins. Notably, EOM fibers express a diverse array of myosin heavy-chain isoforms, retaining embryonic forms into adulthood. Moreover, their motor innervation is characterized by a high ratio of nerve fibers to muscle fibers and the presence of unique neuromuscular junctions. These features contribute to the specialized functions of EOMs, including rapid and precise eye movements. Understanding the mechanisms behind the resilience of EOMs to disease and aging may offer insights into potential therapeutic strategies for treating muscular dystrophies and myopathies affecting other skeletal muscles.

Predicting Myosin Heavy Chain Isoform Through Muscle Fiber Length in Older Adults

Myosin heavy chain (MHC) isoforms directly impact muscle cellular contractile characteristics and are closely associated with other cellular functions (substrate metabolism, calcium handling, etc.). As such, MHC isoform has come to define muscle “fiber type” (MHC I = slow/oxidative and MHC II = fast/glycolytic). Due to the heterogeneity of MHC isoforms expressed within human skeletal muscle tissue, it is essential to determine MHC isoform to allow proper interpretation of tissue level outcomes that may arise from variations within or between fiber types. A priori estimation of fiber type would be a useful tool for ensuring even distribution of fiber type when considering cellular assays in which MHC isoform can only be determined after the fact. Based on the assumption that fibers of various MHC isoform would differently resist mechanical strain, we’ve demonstrated that MHC I fibers can be distinguished from MHC II fibers based on the ratio of fiber to bundle length (degree of stretch) during mechanical dissection. However, our approach used samples from healthy young adults and has not been tested in tissue from older adults, where cellular stiffness is likely elevated. Therefore, our objective is to test our ability to predict MHC isoform in older adults. We hypothesize that muscle fiber strain following dissection will predict MHC isoform in older adults. Methods: Muscle biopsies from the vastus lateralis muscle were obtained from two older adults (one male, one female). A total of 129 muscle fibers were dissected and measured. Fibers were then stored individually and MHC isoform was subsequently identified via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page). Receiver Operating Characteristics (ROC) and confusion matrix were used to assess the predictive capacity for fiber stretch on activation as an index of fiber type. Data: An ROC area under curve (AUC) of 0.634 which indicates that there is a relatively low predictive capacity. Summary: This study aimed to test the feasibility of an approach for predicting muscle fiber type through changes in fiber length upon mechanical dissection. Established in young adults, this study tested samples obtained from older adults to explore the potential effects of age. Preliminary data show that muscle fiber length can predict muscle fiber type in older adults, though the accuracy may be limited when compared with the same approach in younger adults. This lack of accuracy may be attributed to the increase in stiffness that occurs with age, limiting the capacity for strain and thus fiber type variability. A new threshold may need to be determined to increase the accuracy of fiber type prediction. Conclusion: Preliminary data shows that fiber length can provide a prediction for MHC isoform in older adults. NIH National Institute on Aging (1R21AG077125-01A1) and Achievement Rewards for College Scientists (ARCS) Oregon. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.