MHC Isoforms Research Articles

Supra-physiological dose of testosterone induces pathological cardiac hypertrophy.

Testosterone and androgenic anabolic steroids have been misused for enhancement of physical performance despite many reports on cardiac sudden death. Although physiological level of testosterone provided many regulatory benefits to human health, including the cardiovascular function, supra-physiological levels of the hormone induce hypertrophy of the heart with unclear contractile activation. In this study, dose- and time-dependent effects of high-testosterone treatment on cardiac structure and function were evaluated. Adult male rats were divided into four groups of testosterone treatment for 0, 5, 10, and 20 mg/kg BW for 4, 8, or 12 weeks. Increases in both percentage heart:body weight ratio and cardiomyocyte cross-sectional area in representing hypertrophy of the heart were significantly shown in all testosterone-treated groups to the same degree. In 4-week-treated rats, physiological cardiac hypertrophy was apparent with an upregulation of α-MHC without any change in myofilament contractile activation. In contrast, pathological cardiac hypertrophy was observed in 8- and 12-week testosterone-treated groups, as indicated by suppression of myofilament activation and myocardial collagen deposition without transition of MHC isoforms. Only in 12-week testosterone-treated group, eccentric cardiac hypertrophy was demonstrated with unaltered myocardial stiffness, but significant reductions in the phosphorylation signals of ERK1/2 and mTOR. Results of our study suggest that the outcome of testosterone-induced cardiac hypertrophy is not dose dependent but is rather relied on the factor of exposure to duration in inducing maladaptive responses of the heart.

Crossbridge Recruitment Dynamics are Divergently Affected by α/β-Myosin Heavy Chain Isoforms in Cardiac Muscle Fibers Containing the Hypertrophic Cardiomyopathy Mutation (A30V) and Protein Kinase C Phosphomimetic (T203E) in Mouse Cardiac Troponin T

The hypertrophic cardiomyopathy (HCM) mutation A30V is located within the N-terminus of troponin T (TnT), a region known to regulate Ca2+-activated maximal tension and length-dependent activation (LDA) in cardiac muscle. Given how the function of the N-terminus is differently altered by α/β-myosin heavy chain (MHC) isoforms, we believe the mutation-mediated effects are also differently altered by α- and β-MHC isoforms. Likewise, the effects of protein kinase C (PKC)-mediated phosphorylation of TnT (residue T203) are also differently altered by MHC isoforms; phosphorylation of TnT is important to consider because PKC isoforms are upregulated in failing human hearts. We tested the effects of TnTA30V (mouse analog of human A28V), PKC phosphomimetic T203E (TnTT203E), and TnTT203E+A30V in α- and β-MHC backgrounds. In α-MHC fibers, Ca2+-activated maximal tension was attenuated by TnTA30V (∼11%) and TnTT203E+A30V (∼59%). However, in β-MHC fibers, maximal tension was augmented by TnTA30V (∼13%), but was attenuated by TnTT203E+A30V (∼29%) to a lesser extent than in α-MHC fibers. Rate constants of tension redevelopment (ktr) and crossbridge (XB) recruitment dynamics (b) were attenuated by TnTT203E+A30V in α-MHC fibers, but were unaffected in β-MHC fibers. The magnitude of muscle length-mediated recruitment of strong XBs (ER) was attenuated by TnTA30V (∼31%) and TnTT203E+A30V (∼43%) in α-MHC fibers. In contrast, ER was augmented by TnTA30V (∼65%) and TnTT203E+A30V (∼65%) in β-MHC fibers. Inferences drawn from dynamic and steady-state contractile measurements suggest that XB-RU cooperativity - a significant player in LDA - is attenuated by TnTA30V and TnTT203E+A30V in α-MHC, but augmented in β-MHC. These observations demonstrate that α/β-MHC isoforms divergently modify contractile dynamics in TnTA30V and TnTT203E+A30V fibers.

Exercise preconditioning prevents MCT-induced right ventricle remodeling through the regulation of TNF superfamily cytokines

BackgroundExercise training has been recognized as a non-pharmacological therapeutic approach in several chronic diseases; however it remains to be tested if exercise preconditioning can positively interfere with the natural history of pulmonary arterial hypertension (PAH). This is important since the majority of these patients are diagnosed at advanced stages of the disease, when right ventricle (RV) impairment is already present. ObjectivesIn the current study, we evaluated the preventive effect of exercise preconditioning on RV failure secondary to PAH, with a focus on the signaling pathways modulated by pro-inflammatory cytokines from TNF superfamily. MethodsWe analyzed the RV muscle from adult male Wistar rats exposed to a 4-week treadmill exercise training or sedentary regime, prior to the administration of monocrotaline (MCT) to induce PAH or with saline solution (controls). ResultsData indicate that exercise preconditioning prevented cardiac hypertrophy and RV diastolic dysfunction. At a molecular level, exercise modulated the TWEAK/NF-κB signaling axis and prevented the shift in MHC isoforms towards an increased expression of beta-MHC. Exercise preconditioning also prevented the increase of atrogin-1 expression, and induced a shift of MMP activity from MMP-9 to MMP-2 activity. ConclusionsAltogether, data support exercise as a preventive strategy for the management of PAH, which is of particular relevance for the familial form of PAH that is manifested by greater severity or earlier onset.

Naringenin modulates skeletal muscle differentiation via estrogen receptor α and β signal pathway regulation.

Several experiments sustain healthful benefits of the flavanone naringenin (Nar) against chronic diseases including its protective effects against estrogen-related cancers. These experiments encourage Nar use in replacing estrogen treatment in post-menopausal women avoiding the serious side effects ascribed to this hormone. However, at the present, scarce data are available on the impact of Nar on E2-regulated cell functions. This study was aimed at determining the impact of Nar on the estrogen receptor (ERα and β)-dependent signals important for 17β-estradiol (E2) effect in muscle cells (rat L6 myoblasts, mouse C2C12 myoblasts, and mouse skeletal muscle satellite cells). Dietary relevant concentration of Nar delays the appearance of skeletal muscle differentiation markers (i.e., GLUT4 translocation, myogenin, and both fetal and slow MHC isoforms) and impairs E2 effects specifically hampering ERα ability to activate AKT. Intriguingly, Nar effects are specific for E2-initiating signals because IGF-I-induced AKT activation, and myoblast differentiation markers were not affected by Nar treatment. Only 7days after Nar stimulation, early myoblast differentiation markers (i.e., myogenin, and fetal MHC) start to be accumulated in myoblasts. On the other hand, Nar stimulation activates, via ERβ, the phosphorylation of p38/MAPK involved in reducing the reactive oxygen species formation in skeletal muscle cells. As a whole, data reported here strongly sustain that although Nar action mechanisms include the impairment of ERα signals which drive muscle cells to differentiation, the effects triggered by Nar in the presence of ERβ could balance this negative effect avoiding the toxic effects produced by oxidative stress .

Gene expression in mdx mouse muscle in relation to age and exercise: aberrant mechanical–metabolic coupling and implications for pre-clinical studies in Duchenne muscular dystrophy

Weakness and fatigability are typical features of Duchenne muscular dystrophy patients and are aggravated in dystrophic mdx mice by chronic treadmill exercise. Mechanical activity modulates gene expression and muscle plasticity. Here, we investigated the outcome of 4 (T4, 8 weeks of age) and 12 (T12, 16 weeks of age) weeks of either exercise or cage-based activity on a large set of genes in the gastrocnemius muscle of mdx and wild-type (WT) mice using quantitative real-time PCR. Basal expression of the exercise-sensitive genes peroxisome-proliferator receptor γ coactivator 1α (Pgc-1α) and Sirtuin1 (Sirt1) was higher in mdx versus WT mice at both ages. Exercise increased Pgc-1α expression in WT mice; Pgc-1α was downregulated by T12 exercise in mdx muscles, along with Sirt1, Pparγ and the autophagy marker Bnip3. Sixteen weeks old mdx mice showed a basal overexpression of the slow Mhc1 isoform and Serca2; T12 exercise fully contrasted this basal adaptation as well as the high expression of follistatin and myogenin. Conversely, T12 exercise was ineffective in WT mice. Damage-related genes such as gp91-phox (NADPH-oxidase2), Tgfβ, Tnfα and c-Src tyrosine kinase were overexpressed in mdx muscles and not affected by exercise. Likewise, the anti-inflammatory adiponectin was lower in T12-exercised mdx muscles. Chronic exercise with minor adaptive effects in WT muscles leads to maladaptation in mdx muscles with a disequilibrium between protective and damaging signals. Increased understanding of the pathways involved in the altered mechanical-metabolic coupling may help guide appropriate physical therapies while better addressing pharmacological interventions in translational research.

Changes in αB-crystallin, tubulin, and MHC isoforms by hindlimb unloading show different expression patterns in various hindlimb muscles.

[Purpose]αB-crystallin is a small heat shock protein that acts as a molecular chaperone under various stress conditions. Microtubules, which consist of tubulin, are related to maintain the intracellular organelles and cellular morphology. These two proteins have been shown to be related to the properties of different types of myofibers based on their contractile properties. The response of these proteins during muscular atrophy, which induces a myofibril component change, is not clearly understood.[Methods]We performed 15 days of hindlimb unloading on rats to investigate the transitions of these proteins by analyzing their absolute quantities. Protein contents were analyzed in the soleus, plantaris, and gastrocnemius muscles of the unloading and control groups (N = 6).[Results]All three muscles were significantly atrophied by hindlimb unloading (P < 0.01): soleus (47.5%), plantaris (16.3%), and gastrocnemius (21.3%) compared to each control group. αB-crystallin was significantly reduced in all three examined unloaded hindlimb muscles compared to controls (P < 0.01) during the transition of the myosin heavy chain to fast twitch muscles. α-Tubulin responded only in the unloaded soleus muscle. Muscle atrophy induced the reduction of αB-crystallin and α-tubulin expressions in plantar flexor muscles with a shift to the fast muscle fiber compared to the control.[Conclusion]The novel finding of this study is that both proteins, αB-crystallin and α-tubulin, were downregulated in slow muscles (P < 0.01); However, α-tubulin was not significantly reduced compared to the control in fast muscles (P < 0.01).

Gene expression and β-adrenergic signaling are altered in hypoplastic left heart syndrome

The purpose of the current study was to define the myocellular changes and adaptation of the β-adrenergic receptor (β-AR) system that occur in the systemic right ventricle (RV) of children with hypoplastic left heart syndrome (HLHS). Explanted hearts from children with HLHS and non-failing controls were used for this study. HLHS patients were divided into 2 groups: "compensated" (C-HLHS), infants listed for primary transplant with normal RV function and absence of heart failure symptoms, and "decompensated" (D-HLHS), patients listed for transplant after failed surgical palliation with RV failure and/or refractory protein-losing enteropathy or plastic bronchitis. Compared with non-failing control RVs, the HLHS RV demonstrated decreased sarcoplasmic reticulum calcium-adenosine triphosphatase 2a and α-myosin heavy chain (MHC) gene expression, decreased total β-AR due to down-regulation of β1-AR, preserved cyclic adenosine monophosphate levels, and increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity. There was increased atrial natriuretic peptide expression only in the C-HLHS group. Unique to those in the D-HLHS group was increased β-MHC and decreased α-MHC protein expression (MHC isoform switching), increased adenylyl cyclase 5 expression, and increased phosphorylation of the CaMK target site on phospholamban, threonine 17. The HLHS RV has an abnormal myocardial gene expression pattern, downregulation of β1-AR, preserved cyclic adenosine monophosphate levels, and increased CaMKII activity compared with the non-failing control RV. There is MHC isoform switching, increased adenylyl cyclase 5, and increased phosphorylation of phospholamban threonine 17 only in the D-HLHS group. Although abnormal gene expression and changes in the β-AR system precede clinically evident ventricular failure in HLHS, additional unique adaptations occur in those with HLHS and failed surgical palliation.

Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Exercise training induced myosin heavy chain isoform alteration in the infarcted heart

The myosin heavy chain isoform MHC-α has 3-fold higher ATPase activity than MHC-β. After myocardial infarction (MI), MHC-α expression is profoundly downregulated and MHC-β expression is reciprocally upregulated. This shift, which is attributed to low thyroid hormone (TH), contributes to myocardial systolic dysfunction. We investigated the effect of post-MI exercise training on MHC isoforms, TH, and cardiac function. MI was surgically induced in 7-week-old rats by ligation of the coronary artery. The survivors were assigned to 3 groups (n = 10/group): Sham (no MI, no exercise), MISed (MI, no exercise), and MIEx (MI, exercise). Treadmill exercise training began 1 week post-MI and lasted for 8 weeks. Echocardiogram measurements were taken on the day prior to initiation of exercise training and at the end of exercise training. Tissue and blood samples were collected at the end of the experiment. MHC isoform gene and protein expression and TH were measured. Our results illustrated that MHC-α gene expression was higher and MHC-β gene expression was lower in the MIEx group than in the MISed group. Resting serum TH concentrations (T3 and T4) were similar between the 2 MI groups. The MIEx group had higher fractional shortening than the MISed group. In conclusion, post-MI exercise training beneficially altered MHC isoforms and improved cardiac function without changing TH.

Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [14C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.

Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and β-Tm) and slow (γ-Tm and β-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, β and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Effect of Estrogen on Molecular and Functional Characteristics of the Rodent Vaginal Muscularis

Vaginal atrophy is a consequence of menopause; however, little is known concerning the effect of a decrease in systemic estrogen on vaginal smooth muscle structure and function. As the incidence of pelvic floor disorders increases with age, it is important to determine if estrogen regulates the molecular composition and contractility of the vaginal muscularis. The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis utilizing a rodent model of surgical menopause. Three- to 4-month old Sprague-Dawley rats underwent sham laparotomy (Sham, N = 18) or ovariectomy (Ovx, N = 39). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17β-estradiol (Ovx). Animals were euthanized 1 week later, and the proximal vagina was collected for analysis of contractile protein expression and in vitro studies of contractility. Measurements were analyzed using a one-way analysis of variance followed by Tukey's post hoc analysis (α = 0.05). Protein and mRNA transcript expression levels of contractile proteins, in vitro measurements of vaginal contractility. Ovariectomy decreased the expression of carboxyl-terminal myosin heavy chain isoform (SM1) and h-caldesmon and reduced the amplitude of contraction of the vaginal muscularis in response to KCl. Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, mRNA transcript expression of amino-terminal MHC isoforms, l-caldesmon expression, and maximal velocity of shortening. Systemic estrogen replacement restores functional and molecular characteristics of the vaginal muscularis of ovariectomized rats. Our results indicate that menopause is associated with changes in the vaginal muscularis, which may contribute to the increased incidence of pelvic floor disorders with age.

Satellite cell pool expansion is affected by skeletal muscle characteristics

We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. Participants completed a downhill running (DHR) intervention (5 × 8 min, 2-min rest; 80% VO2max ; -10% gradient). Muscle biopsies were taken 7 days before VO₂max and 7-9 days after the DHR intervention. Delayed-onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post-DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. DOMS and CK peaked on day 1 post-DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO₂max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7(+) cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO₂max .

The contribution of stem cell therapy to skeletal muscle remodeling in heart failure

The aim of our study was to investigate whether stem cell (SC) therapy with human amniotic fluid stem cells (hAFS, fetal stem cells) and rat adipose tissue stromal vascular fraction cells-GFP positive cells (rSVC-GFP) was able to produce favorable effects on skeletal muscle (SM) remodeling in a well-established rat model of right heart failure (RHF). RHF was induced by monocrotaline (MCT) in Sprague-Dawley rats. Three weeks later, four millions of hAFS or rSVC-GFP cells were injected via tail vein. SM remodeling was assessed by Soleus muscle fiber cross sectional area (CSA), myocyte apoptosis, myosin heavy chain (MHC) composition, satellite cells pattern, and SC immunohistochemistry. hAFS and rSVC-GFP injection produced significant SC homing in Soleus (0.68 ± 1.0 and 0.67 ± 0.75% respectively), with a 50% differentiation toward smooth muscle and endothelial cells. Pro-inflammatory cytokines were down regulated to levels similar to those of controls. SC-treated (SCT) rats showed increased CSA (p<0.004 vs MCT) similarly to controls with a reshift toward the slow MHC1 isoform. Apoptosis was significantly decreased (11.12.± 8.8 cells/mm(3) hAFS and 13.1+7.6 rSVC-GFP) (p<0.001 vs MCT) and similar to controls (5.38 ± 3.0 cells/mm(3)). RHF rats showed a dramatic reduction of satellite cells(MCT 0.2 ± 0.06% Pax7 native vs controls 2.60 ± 2.46%, p<0.001), while SCT induced a repopulation of both native and SC derived satellite cells (p<0.005). SC treatment led to SM remodeling with satellite cell repopulation, decreased atrophy and apoptosis. Modulation of the cytokine milieu might play a crucial pathophysiological role with a possible scenario for autologous transplantation of SC in pts with CHF myopathy.

Effects of the N-Terminus of Cardiac Troponin T on Contractile Activation Depend on the Type of Myosin Heavy Chain Isoform

We have previously shown that two specific regions (residues 1-43 and residues 44-73) in rat cardiac troponin T (RcTnT) have divergent effects on contractile function against an α-myosin heavy chain (α-MHC) background (Mamidi et al., Abstract: Biophys J, vol.100, Issue 3, p586a, 2010). Because the effects of Tn isoforms are modulated by an interplay with MHC, we hypothesized that the divergent effects of residues 1-43 and residues 44-73 in cTnT would be further modulated by a shift in MHC isoform to β-MHC. This has a direct bearing on understanding the effects of alterations in cTnT and MHC isoforms not only in health but also in disease. We generated two recombinant mutants (RcTnT1-43 and RcTnT44-73) by deleting residues 1-43 and 44-73 in RcTnT, respectively. We studied contractile function by reconstituting RcTnT1-43 and RcTnT44-73 recombinant proteins into detergent-skinned papillary muscle fibers from normal rat hearts (α-MHC) and propylthiouracil-treated rat hearts (β-MHC). Our data shows that functional alterations induced by RcTnT1-43 and RcTnT44-73 were different in α-MHC vs. β-MHC fibers. For example, RcTnT1-43 decreased both Ca2+-activated maximal tension (by ∼46%) and myofilament Ca2+-sensitivity (by 0.09 pCa units) in α-MHC fibers. However, RcTnT1-43 modestly decreased maximal tension (by ∼18%), with no effect on Ca2+-sensitivity in β-MHC fibers. Thus, the desensitizing effect of RcTnT1-43 was attenuated by β-MHC. Ca2+-activated maximal tension data were supported by myofiber dynamic stiffness measurements. Another major finding is that RcTnT44-73 abolished the sarcomere length-dependent increase in Ca2+-sensitivity in β-MHC, but not in α-MHC fibers. Thus, our data demonstrates that the functional outcome of the N-terminus of cTnT is modulated by the type of MHC isoform present.

Four days of muscle disuse impairs single fiber contractile function in young and old healthy men

The purpose of the study was to investigate the effects of 4days of disuse (knee brace) on contractile function of isolated vastus lateralis fibers (n=486) from 11 young (24.3±0.9yrs) and 11 old (67.2±1.0yrs) healthy men having comparable levels of physical activity. Prior to disuse single fiber specific force (maximal Ca2+-activated force per cross-sectional area) was lower in MHC I vs. IIa fibers (p<0.05) both in young (44%) and old (32%), and specific force of MHC IIa fibers was lower in old vs. young (19%, p<0.05). Further, Ca2+ sensitivity was higher in MHC I vs. IIa fibers (p<0.05) in both age groups. Following disuse single fiber specific force decreased (p<0.05) in MHC I fibers in young (−19%) and old (−17%), and in MHC IIa fibers in young (−21%) but not in old (−11%; ns). The extent of these decreases did not differ between young and old. Ca2+ sensitivity decreased (p<0.05) in MHC I fibers in old (−0.11 pCa units) but not in young (−0.08 pCa units; ns). The extent of these decreases was greater in old vs. young (p<0.05). In conclusion, 4days of lower limb disuse led to marked impairments in single muscle fiber specific force independently of age, while changes in Ca2+ sensitivity were dependent on age and MHC isoform composition. The present findings stress the importance of determining and implementing effective preventive and rehabilitative approaches for old individuals exposed to as little as 4days of disuse.

Myosin Isoform Fiber Type and Fiber Size in the Tail of the Virginia Opossum (Didelphis virginiana)

Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function.

Age-related changes in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins, and subneural apparatuses

We compared age-related changes in the intrinsic laryngeal muscles of aged and young adult rats by determining the number and diameter of muscle fibers, contractile muscle protein (myosin heavy chain isoforms, MHC) composition, and the morphology of the subneural apparatuses. In aged rats, both the numbers and the diameters of muscle fibers decreased in the cricothyroid (CT) muscle. The number of fibers, but not diameter, decreased in the thyroarytenoid (TA) muscle. In the posterior cricoarytenoid (PCA) muscle, neither the number nor the diameter of fibers changed significantly. Aging was associated with a decrease in type IIB and an increase in type IIA MHC isoform levels in CT muscle, but no such changes were observed in the TA or PCA muscles. Morphological examination of primary synaptic clefts of the subneural apparatus revealed that aging resulted in decreased labyrinthine and increased depression types in only the CT muscle. In the aged group, morphologically immature subneural apparatuses were found infrequently in the CT muscle, indicating continued tissue remodeling. We suggest, therefore, that age-related changes in the intrinsic laryngeal muscles primarily involve the CT muscle, whereas the structures of the TA and PCA muscles may better resist aging processes and therefore are less vulnerable to functional impairment. This may reflect differences in their roles; the CT muscle controls the tone of the vocal folds, while the TA and PCA muscles play an essential role in vital activities such as respiration and swallowing.

Effects of R92 mutations in mouse cardiac troponin T are influenced by changes in myosin heavy chain isoform

One limitation in understanding how different familial hypertrophic cardiomyopathy (FHC)-related mutations lead to divergent cardiac phenotypes is that such mutations are often studied in transgenic (TG) mouse hearts which contain a fast cycling myosin heavy chain isoform (α-MHC). However, the human heart contains a slow cycling MHC isoform (β-MHC). Given the physiological significance of MHC-troponin interplay effects on cardiac contractile function, we hypothesized that cardiac troponin T (cTnT) mutation-mediated effects on contractile function depend on the type of MHC isoform present in the sarcomere. We tested our hypothesis using two variants of cTnT containing mutations at FHC hotspot R92 (R92L or R92Q), expressed against either an α-MHC or β-MHC background in TG mouse hearts. One finding from our study was that R92L attenuated the length-dependent increase in tension and abolished the length-dependent increase in myofilament Ca2+ sensitivity only when β-MHC was present. In addition, α- and β-MHC isoforms differentially affected how R92 mutations altered crossbridge (XB) recruitment dynamics. For example, the rate of XB recruitment was faster in R92L or R92Q fibers when β-MHC was present, but was unaffected when α-MHC was present. The R92Q mutation sped XB detachment in the presence of β-MHC, but not in the presence of α-MHC. R92Q affected the XB strain-dependent influence on XB recruitment dynamics, an effect not observed for R92L. Our findings have major implications for understanding not only the divergent effects of R92 mutations on cardiac phenotype, but also the distinct effects of MHC isoforms in determining the outcome of mutations in cTnT.

Human skeletal muscle biochemical diversity

The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.