Changes In Myosin Heavy Chain Isoforms Research Articles

Diet and Exercise Restores Muscle Fiber Phenotype Following Long Term High Fat/High Sugar Diet Yet mRNA Expression Remains Unchanged

Obesity affects roughly 42% of the US population. High fat/high sugar diets (HFHS) often referred as “western diet” contributes to this prevalence. Diet‐induced obesity (DIO) results in impaired metabolic responses and associated disease states (i.e., Type 2 Diabetes). Metabolic impairments in DIO are a result of changes in muscle metabolism and muscle fiber phenotype. This is determined by both mRNA expression and protein isoform‐content of myosin heavy chain (MHC). Fast muscle fiber phenotype (i.e., type IIb in mice) is characterized by lower capacity for lipid utilization, implicated in the pathogenesis of Type 2 Diabetes. Regular exercise expresses mRNA in a fiber type specific manner, shifting MHC proportions under healthy circumstances. However, diet and exercise‐driven fiber type shifts in DIO are less understood. We aimed to determine the impact of exercise and diet on fiber‐type proportions in mice with DIO. We hypothesized exercise shifts mouse gastroc muscle phenotype induced by HFHS diet away from IIb fast fiber types. 49 C57BL/6 mice were split into 4 groups: 1) Lean Control (n = 9) fed standard chow and water for 24 wks, 2) HFHS (n = 10), fed HFHS diet (60% of calories from fat, high sugar/fructose: 42 g/L in drinking water) for 24 wks, 3) HFHS Control (n = 10) fed HFHS diet for 12 wks followed by standard chow and water for 12 wks (i.e., simulating traditional diet approach), and 4) HFHS + exercise group (n = 10) fed HFHS diet for 24 wks and performed aerobic exercise (30 min of treadmill running 5 d/wk) in the last 12 wks. Gastroc muscles were collected, homogenized, and analyzed for MHC mRNA expression of MYH 1 (type IIx), MYH 2 (type IIa), MYH 4 (type IIb), and protein isoforms via q‐PCR and SDS‐PAGE respectively. Intensity of bands corresponding to MHC isoforms were quantified using Image J. mRNA expression was calculated using comparative CT analysis (2‐∆∆CT). Paired sample t‐tests were conducted to test differences between MHC isoforms across groups. Proportions of MHC IIb isoform increased (91 ± 3%) in HFHS compared to Control (81 ± 6.2%, p>0.05), HFHS Control (77 ± 3%, p=0.004), and HFHS + exercise groups (79 ± 5%, p=0.057). Additionally, MHC IIa/x proportions in the HFHS (8 ± 3%) compared to Control (17 ± 5.9%, p>0.05), HFHS Control (20 ± 2%, p=0.004), and HFHS + exercise (18 ± 4%, p=0.054) groups were reduced. There were no differences in mRNA expression data. These data suggest performing aerobic exercise with HFHS diet or switching to a standard diet restores muscle fiber phenotype in mouse gastroc. Yet, this was not reflected in mRNA expression data. Changes in MHC isoforms are likely better detected at the protein level rather than mRNA expression. We conclude that exercise and dietary intervention may be a good strategy to shift MHC isoforms away from extreme fast fiber phenotype with lower capacity for lipid utilization. Future research should aim to determine single muscle fiber phenotype shifts with long‐term diet and exercise in humans with obesity to better understand regulation of muscle fiber phenotypes impact on human metabolism.

All-trans retinoic acid increases the expression of oxidative myosin heavy chain through the PPARδ pathway in bovine muscle cells derived from satellite cells.

All-trans retinoic acid (ATRA) has been associated with various physiological phenomenon in mammalian adipose tissue and skeletal muscle. We hypothesized that ATRA may affect skeletal muscle fiber type in bovine satellite cell culture through various transcriptional processes. Bovine primary satellite cell (BSC) culture experiments were conducted to determine dose effects of ATRA on expression of genes and protein levels related to skeletal muscle fiber type and metabolism. The semimembranosus from crossbred steers (n = 2 steers), aged approximately 24 mo, were used to isolate BSC for 3 separate assays. Myogenic differentiation was induced using 3% horse serum upon cultured BSC with increasing doses (0, 1, 10, 100, and 1,000 nM) of ATRA. After 96 h of incubation, cells were harvested and used to measure the gene expression of protein kinase B (Akt), AMP-activated protein kinase alpha (AMPK), glucose transporter 4 (GLUT4), myogenin, lipoprotein lipase (LPL), myosin heavy chain (MHC) I, MHC IIA, MHC IIX, insulin like growth factor-1 (IGF-1), Peroxisome proliferator activated receptor gamma (PPARγ), PPARδ, and Smad transcription factor 3 (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). The mRNA expression of LPL was increased (P < 0.05) with 100 and 1,000 nM of ATRA. Expression of GLUT4 was altered (P < 0.05) by ATRA. The treatment of ATRA (1,000 nM) also increased (P < 0.05) mRNA gene expression of SMAD3. The gene expression of both PPARδ and PPARγ were increased (P < 0.05) with 1,000 nM of ATRA. Protein level of PPARδ was also affected (P < 0.05) by 1,000 nM of ATRA and resulted in a greater (P < 0.05) protein level of PPARδ compared to CON. All-trans retinoic acid (10 nM) increased gene expression of MHC I (P < 0.05) compared to CON. Expression of MHC IIA was also influenced (P < 0.05) by ATRA. The mRNA expression of MHC IIX was decreased (P < 0.05) with 100 and 1,000 nM of ATRA. In muscle cells, ATRA may cause muscle fibers to transition towards the MHC isoform that prefers oxidative metabolism, as evidenced by increased expression of genes associated with the MHC I isoform. These changes in MHC isoforms appeared to be brought about by changing PPARδ gene expression and protein levels.

Polar bears experience skeletal muscle atrophy in response to food deprivation and reduced activity in winter and summer.

When reducing activity and using stored energy during seasonal food shortages, animals risk degradation of skeletal muscles, although some species avoid or minimize the resulting atrophy while experiencing these conditions during hibernation. Polar bears may be food deprived and relatively inactive during winter (when pregnant females hibernate and hunting success declines for other demographic groups) as well as summer (when sea ice retreats from key foraging habitats). We investigated muscle atrophy in samples of biceps femoris collected from free-ranging polar bears in the Southern Beaufort Sea (SBS) throughout their annual cycle. Atrophy was most pronounced in April-May as a result of food deprivation during the previous winter, with muscles exhibiting reduced protein concentration, increased water content, and lower creatine kinase mRNA. These animals increased feeding and activity in spring (when seal prey becomes more available), initiating a period of muscle recovery. During the following ice melt of late summer, ~30% of SBS bears abandon retreating sea ice for land; in August, these 'shore' bears exhibited no muscle atrophy, indicating that they had fully recovered from winter food deprivation. These individuals subsequently scavenged whale carcasses deposited by humans and by October, had retained good muscle condition. In contrast, ~70% of SBS bears follow the ice north in late summer, into deep water with less prey. These 'ice' bears fast; by October, they exhibited muscle protein loss and rapid changes in myosin heavy-chain isoforms in response to reduced activity. These findings indicate that, unlike other bears during winter hibernation, polar bears without food in summer cannot mitigate atrophy. Consequently, prolonged summer fasting resulting from climate change-induced ice loss creates a risk of greater muscle atrophy and reduced abilities to travel and hunt.

Myosin Heavy Chain Isoform Switching in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). Since the impact of HCM c/ssTnC mutations during skeletal muscle development and function is unknown, we addressed whether changes in the myosin heavy chain (MHC) isoforms can occur outside of the heart. We hypothesized that the ratio of MHC I to MHC II in various skeletal muscles change due to an HCM-associated c/ssTnC A8V mutation in a knock-in homozygous mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in diaphragm (D), soleus (S), tibialis anterior (TA), extensor digitorum longus (EDL) and quadriceps (Q) isolated from 3- and 9-month (mo) old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE. The muscle/body weight ratio (M/BW) and running capacity were also measured. At 3 mo, MHC isoform switching was observed in D (increased MHCIIb), S (decreased MHCIIa, increased MHCI) and EDL (increased MHCIIa/x, decreased MHCIIb) of KI-TnC-A8V+/+ compared to WT. TA M/BW is increased in 3- and 9-mo KI-TnC-A8V+/+ mice compared to WT at the same age. Pairwise comparison of M/BW from 3- vs 9-mo KI-TnC-A8V+/+ mice showed a significant decrease for TA and Q and an increase for EDL, and no change for any WT muscles. In running tests, KI-TnC-A8V+/+ mice had a lower capacity to run distances compared to WT (281 vs 413 meters). Thus, changes in MHC isoforms in the skeletal muscles of mice expressing c/ssTnC A8V are associated with reduced exercise endurance, suggesting that skeletal muscle remodeling may contribute to the development of HCM phenotype. Future studies will examine MHC isoforms at 9 mo and Ca2+-activated myofibrilar Mg2+-ATPase rates from skeletal muscles in KI-TnC-A8V+/+ mice.

Functional consequence of mutation in rat cardiac troponin T is affected differently by myosin heavy chain isoforms

Cardiac troponin T (cTnT) is an essential component of the thin filament regulatory unit (RU) that regulates Ca2+ activation of tension in the heart muscle. Because there is coupling between the RU and myosin crossbridges, the functional outcome of cardiomyopathy-related mutations in cTnT may be modified by the type of myosin heavy chain (MHC) isoform. Ca2+ activation of tension and ATPase activity were measured in muscle fibres from normal rat hearts containing alpha-MHC isoform and propylthiouracil (PTU)-treated rat hearts containing beta-MHC isoform. Muscle fibres from normal and PTU-treated rat hearts were reconstituted with two different mutations in rat cTnT; the deletion of Glu162 (cTnT(E162DEL)) and the deletion of Lys211 (cTnT(K211DEL)). Alpha-MHC and beta-MHC isoforms had contrasting impact on tension-dependent ATP consumption (tension cost) in cTnT(E162DEL) and cTnT(K211DEL) reconstituted muscle fibres. Significant increases in tension cost in alpha-MHC-containing muscle fibres corresponded to 17% (P < 0.01) and 23% (P < 0.001) when reconstituted with cTnT(E162DEL) and cTnT(K211DEL), respectively. In contrast, tension cost decreased when these two cTnT mutants were reconstituted in muscle fibres containing beta-MHC; by approximately 24% (P < 0.05) when reconstituted with cTnT(E162DEL) and by approximately 17% (P = 0.09) when reconstituted with cTnT(K211DEL). Such differences in tension cost were substantiated by the mechano-dynamic analysis of cTnT mutant reconstituted muscle fibres from normal and PTU-treated rat hearts. Our observation demonstrates that qualitative changes in MHC isoform alters the nature of cardiac myofilament dysfunction induced by mutations in cTnT.

Temporal Response of Desmin and Dystrophin to Progressive Resistance Exercise

PURPOSE: Our purpose was to investigate the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 weeks of progressive resistance training and following a single bout of unaccustomed resistance exercise. METHODS: Muscle biopsies were taken from the vastus lateralis of 12 untrained men a week prior to exercise (week 0) and at weeks 1,2,4, and 8 following a single bout of exercise (n = 6), or at weeks 1, 2, 4 and 8 of the progressive resistance training program (n = 6). Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS PAGE at each time point for each group. RESULTS: In the training group, the desmin:actin ratio significantly increased compared to week 0 beginning at week 4 (0.054 to 0.095, P < 0.0001) and continuing through week 8 (0.054 to 0.094, P < 0.0001). The desmin:actin ratio did not change at any time point for the single bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5719+/−382 and 6582+/−640, weeks 0 and 4 respectively, P < 0.05). Finally, a significant negative correlation was observed between the desmin:actin ratio and the percentage of MHC IIx (R = −0.31, P < 0.05, all time points from both groups). CONCLUSION: These data suggest a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but prior to changes in MHC isoforms, while dystrophin remains unchanged.

Temporal response of desmin and dystrophin proteins to progressive resistance exercise in human skeletal muscle

We have investigated the adaptations of the cytoskeletal proteins desmin and dystrophin in relationship to known muscular adaptations of resistance exercise. We measured desmin, dystrophin, and actin protein contents, myosin heavy chain (MHC) isoform distribution, muscle strength, and muscle cross-sectional area (CSA) during 8 wk of progressive resistance training or after a single bout of unaccustomed resistance exercise. Muscle biopsies were taken from the vastus lateralis of 12 untrained men. For the single-bout group (n=6) biopsies were taken 1 wk before the single bout of exercise (week 0) and 1, 2, 4, and 8 wk after this single bout of exercise. For the training group (n=6), biopsies were taken 1 wk before the beginning of the program (week 0) and at weeks 1, 2, 4, and 8 of the progressive resistance training program. Desmin, dystrophin, and actin protein levels were determined with immunoblotting, and MHC isoform distribution was determined using SDS-PAGE at each time point for each group. In the training group, desmin was significantly increased compared with week 0 beginning at week 4 (182% of week 0; P<0.0001) and remained elevated through week 8 (172% of week 0; P<0.0001). Desmin did not change at any time point for the single-bout group. Actin and dystrophin protein contents were not changed in either group at any time point. The percentage of MHC type IIa increased and MHC type IIx decreased at week 8 in the training group with no changes occurring in the single-bout group. Strength was significantly increased by week 2 (knee extension) and week 4 (leg press), and it further increased at week 8 for both these exercises in the training group only. Muscle CSA was significantly increased at week 4 for type II fibers in the training group only (5,719+/-382 and 6,582+/-640 microm2, weeks 0 and 4, respectively; P<0.05). Finally, a significant negative correlation was observed between the desmin-to-actin ratio and the percentage of MHC IIx (R=-0.31; P<0.05, all time points from both groups). These data demonstrate a time course for muscular adaptation to resistance training in which desmin increases shortly after strength gains and in conjunction with hypertrophy, but before changes in MHC isoforms, whereas dystrophin remains unchanged.

Myofibrillar myosin ATPase activity in hindlimb muscles from young and aged rats

We tested the hypothesis that Ca 2+-activated myosin ATPase activity is lower in muscles of aged rats relative to muscles of young rats, independent of changes in myosin isoform expression. Myofibrils were prepared from permeabilized fibers of soleus, plantaris, and semimembranosus muscles of young (8–12 months) and aged (32–38 months) F344 × BN rats and assayed for resting myosin ATPase, Ca 2+-activated myosin ATPase, and myosin heavy chain (MHC) and myosin light chain (MLC) isoform compositions. Resting myosin ATPases were not affected by age in any muscle ( P ≥ 0.42). Ca 2+-activated myosin ATPases of soleus and plantaris myofibrils were not affected by age ( P ≥ 0.31) but were 16% lower in semimembranosus myofibrils from aged rats (0.448 ± 0.019 μmol P i/min/mg) compared to young rats (0.533 ± 0.031 μmol P i/min/mg; P = 0.03). Correspondingly, maximal unloaded shortening velocity of single semimembranosus fibers from aged rats was slow (4.6 ± 0.2 fiber lengths/s) compared with fibers from young rats (5.8 ± 0.3 fiber lengths/s; P < 0.01). No age-related changes in MHC or regulatory MLC isoforms were detected in any muscle ( P ≥ 0.08) but changes in the essential MLC occurred in plantaris and semimembranosus muscles. The data indicate that Ca 2+-activated myosin ATPase activity is reduced with age in semimembranosus muscle, independent of age-related changes in MHC isoform expression, and is one mechanism contributing to age-related slowing of contraction in that muscle.

Lack of coordinated changes in metabolic enzymes and myosin heavy chain isoforms in regenerated muscles of trained rats.

We investigated training-induced changes in biochemical properties and myosin heavy chain (MHC) composition of regenerated (cardiotoxin-injected) plantaris muscles (PLA) in rats either maintained sedentary (S, n = 9) or endurance trained on a treadmill over a 8-week period (T, n = 7). Both endurance training and regeneration altered the pattern of fast MHC expression. An analysis of the two-way interaction between training and regeneration showed that the relative content of type IIa MHC was affected (P < 0.05). The 140% increase in type IIa MHC observed in regenerated PLA from T rats compared with nontreated muscle of S rats, exceeded the 102% increase resulting from the combination of regeneration alone (26%) and training alone (61%). A similar interaction between training and regeneration was shown for the percentage of fibres expressing either type IIa or type lIb MHC (P < 0.05). In contrast, a significant increase in the citrate synthase (CS) activity was shown in PLA as a result of endurance training, without specific effect of regeneration. Furthermore, training-induced changes in CK and LDH isoenzyme distribution occurred to a similar extent in regenerated and non-treated PLA muscles, and thus did not follow the changes in MHC isoforms. An increase in the mitochondrial CK isozyme activity (mi-CK) was shown in both non-treated and previously degenerated PLA muscles (123 and 117%, P < 0.01, respectively), without specific effect of regeneration. The ratio of mi-CK to CS activity, an estimate of the mitochondrial specific activity of mi-CK was significantly increased by training (P < 0.02) and decreased by regeneration (P < 0.05). Taken together, these data suggest that while training and regeneration have cumulative effects on the pattern of fast MHC expression, the training-induced changes in the energy metabolism shown in mature non-treated myofibres are similar to those observed in regenerated fibres.

The effect of autografting on the myosin composition in skeletal muscle fibers.

The purpose of the study was to investigate the changes in myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following autotransplantation of extensor digitorum longus muscles. Muscles were grafted in "standard" and "nerve-intact" conditions. MHC and MLC isoforms were analyzed by sodium dodecyl sulphate gel electrophoresis. Changes in MHC isoforms 10, 30, and 60 days after grafting were similar in the "standard" and the "nerve-intact" grafts. In contrast to MHC, changes in MLC were different in the 10th day groups, but the same in the 30th day groups. Sixty days after grafting the content of MLC isoforms was the same as the control muscles. These data indicate that transient loss of functional innervation, even for a short time, has permanent effect on the composition of MHC but not MLC isoforms in regenerating skeletal muscle fibers.

Effects of thyroid hormone on left ventricular performance and regulation of contractile and Ca(2+)-cycling proteins in the baboon. Implications for the force-frequency and relaxation-frequency relationships.

The transcriptional, posttranscriptional, and related functional effects of thyroid hormone on primate myocardium are poorly understood. Therefore, we studied the effects of thyroid hormone on sarcoplasmic reticulum (SR) Ca(2+)-cycling proteins and myosin heavy chain (MHC) composition at the steady state mRNA and protein level and associated alterations of left ventricular (LV) performance in 8 chronically instrumented baboons. The force-frequency and relaxation-frequency relations were assessed as the response of LV isovolumic contraction (dP/dtmax) and relaxation (Tau), respectively, to incremental atrial pacing. Both the heart rate at which dP/dtmax was maximal and Tau was minimal (critical heart rates) in response to pacing were increased significantly after thyroid hormone. Postmortem LV tissue from 5 thyroid-treated and 4 additional control baboons was assayed for steady state mRNA levels with cDNA probes to MHC isoforms and SR Ca(2+)-cycling proteins. Steady state SR Ca(2+)-ATPase and phospholamban mRNA increased in the hyperthyroid state, and alpha-MHC mRNA appeared de novo, whereas beta-MHC mRNA decreased. Western analysis (4 thyroid-treated and 4 control baboons) showed directionally similar changes in MHC isoforms and a slight increase in SR Ca(2+)-ATPase. In contrast, there was a statistically nonsignificant decrease in phospholamban protein, which resulted in a significant 40% decrease in the ratio of phospholamban to SR Ca(2+)-ATPase. Thus, thyroid hormone increases the transcription of Ca(2+)-cycling proteins and shifts MHC isoform expression in the primate LV. Our data suggest that both transcriptional and posttranslational mechanisms determine the levels of these proteins in the hyperthyroid primate heart and mediate, in part, the observed enhanced basal and frequency-dependent LV performance.

Smooth-muscle myosin heavy-chain isoform expression in bladder-outlet obstruction.

Bladder-outlet obstruction leads to detrusor smooth-muscle hypertrophy/hyperplasia. Despite this overall increase in muscle mass, smooth-muscle contractility decreases and bladder emptying is impaired. The goal of this study was to determine whether smooth-muscle myosin heavy-chain (MHC) alterations occur in conjunction with partial obstruction of the rabbit bladder. Total MHC and MHC-isoform protein concentrations were determined by quantitative gel electrophoresis in rabbit bladders partially obstructed for 1-4 weeks. MHC gene expression was assessed by Northern and nuclease protection assays. Two MHC isoforms (SM1/SM2) were identified in the normal rabbit bladder. After 2 weeks of obstruction the ratio of SM1/SM2 changed from 0.4:0.6 to 0.5:0.5 (P < 0.01). As compared with sham-operated values, the level of MHC mRNA decreased significantly as of 1 day after obstruction. Quantitation of MHC-isoform mRNA levels revealed a nearly 3-fold increase in the SM1/SM2 ratio. In this animal model of bladder-outlet obstruction, early changes in MHC isoforms as well as an overall decrease of MHC mRNA expression were demonstrated, suggesting that obstruction induces significant alterations in myofilament gene expression.