Myosin Heavy Chain Isoform Expression Research Articles

Effects of age and diet consistency on the expression of myosin heavy-chain isoforms on jaw-closing and jaw-opening muscles in a rat model.

Skeletal craniofacial morphology can be influenced by changes in masticatory muscle function, which may also change the functional profile of the muscles. To investigate the effects of age and functional demands on the expression of Myosin Heavy-Chain (MyHC) isoforms in representative jaw-closing and jaw-opening muscles, namely the masseter and digastric muscles respectively. Eighty-four male Wistar rats were divided into four age groups, namely an immature (n = 12; 4-week-old), early adult (n = 24; 16-week-old), adult (n = 24; 26-week-old) and mature adult (n = 24; 38-week-old) group. The three adult groups were divided into two subgroups each based on diet consistency; a control group fed a standard (hard) diet, and an experimental group fed a soft diet. Rats were sacrificed, and masseter and digastric muscles dissected. Real-time quantitative polymerase chain reaction was used to compare the mRNA transcripts of the MyHC isoforms-Myh7 (MyHC-I), Myh2 (MyHC-IIa), Myh4 (MyHC-IIb) and Myh1 (MyHC-IIx)-of deep masseter and digastric muscles. In the masseter muscle, hypofunction increases Myh1 (26, 38 weeks; p < .0001) but decreases Myh4 (26 weeks; p = .046) and Myh2 (26 weeks; p < .0001) expression in adult rats. In the digastric muscle, hypofunction increases Myh1 expression in the mature adult rats (38 weeks; p < .0001), while Myh2 expression decreases in adult rats (26 weeks; p = .021) as does Myh4 (26 weeks; p = .001). Myh7 expression is increased in the digastric muscle of mature adult rats subjected to hypofunction (38 weeks; p = <.0001), while it is very weakly expressed in the masseter. In jaw-opening and jaw-closing muscles, differences in myosin expression between hard- and soft-diet-fed rats become evident in adulthood, suggesting that long-term alteration of jaw function is associated with changes in the expression of MyHC isoforms and potential fibre remodelling. This may give insight into the role of function on masticatory muscles and the resultant craniofacial morphology.

Myosin heavy chain isoform expression and meat quality characteristics of different muscles in yak (Bos grunniens)

Myosin heavy chain (MHC) isoforms and meat quality characteristics of different muscles were investigated to explore their potential relationships in yaks. Results showed that semitendinosus (ST), longissimus thoracis (LT), and infraspinatus (IS) have a greater ratio of MHC IIb (47.84%), MHC IIa (73.27%), and MHC I (24.26%), respectively, than the other two muscles. Compared with LT or ST, IS exhibited more intense color, greater water-holding capacity, and initial tenderness with higher intermuscular fat (IMF) and collagen (of lower cross-linking level), presenting overall better quality. Variations in MHC isoforms accounted for the muscle-specific meat quality. Specifically, MHC I was positively associated with redness, myoglobin, IMF, collagen, pH, and thermal stability and negatively associated with myofibril fragmentation index, fiber thickness, collagen cross-linking, and drip loss. These results provide insights into the relationships between MHC isoforms and meat quality in yaks and the MHC I isoform has an extensive influence on meat quality.

Gromwell ameliorates glucocorticoid-induced muscle atrophy through the regulation of Akt/mTOR pathway

BackgroundMuscle atrophy is characterized by decreased muscle mass, function, and strength. Synthetic glucocorticoids, including dexamethasone (Dexa), are commonly used to treat autoimmune diseases. However, prolonged exposure of Dexa with high dose exerts severe side effects, including muscle atrophy. The purpose of this study was to investigate whether Gromwell root extract (GW) can prevent Dexa-induced muscle atrophy in C2C12 cells and mice and to characterize the composition of GW to identify bioactive compounds.MethodsFor in vitro experiments, GW (0.5 and 1 µg/mL) or lithospermic acid (LA, 5 and 10 µM) was added to C2C12 myotubes on day 4 of differentiation and incubated for 24 h, along with 50 µM Dexa. For in vivo experiment, four-week-old male C57BL/6 mice were randomly divided into the four following groups (n = 7/group): Con group, Dexa group, GW0.1 group, and GW0.2 group. Mice were fed experimental diets of AIN-93 M with or without 0.1 or 0.2% GW for 4 weeks. Subsequently, muscle atrophy was induced by administering an intraperitoneal injection of Dexa at a dose of 15 mg/kg/day for 38 days, in conjunction with dietary intake.ResultsIn Dexa-induced myotube atrophy, treatment with GW increased myotube diameter, reduced the expression of muscle atrophy markers, and enhanced the expression of myosin heavy chain (MHC) isoforms in C2C12 cells. Supplementation with the GW improved muscle function and performance in mice with Dexa-induced muscle atrophy, evidenced in the grip strength and running tests. The GW group showed increased lean body mass, skeletal muscle mass, size, and myosin heavy chain isoform expression, along with reduced skeletal muscle atrophy markers in Dexa-injected mice. Supplementation with GW increased protein synthesis and decreased protein degradation through the Akt/mammalian target of rapamycin and glucocorticoid receptor/forkhead box O3 signaling pathways, respectively. We identified LA as a potential bioactive component of the GW. LA treatment increased myotube diameter and decreased the expression of muscle atrophy markers in Dexa-induced C2C12 cells.ConclusionsThese findings underscore the potential of the GW in preventing Dexa-induced skeletal muscle atrophy and highlight the contribution of LA to its effects.

Predicting myosin heavy chain isoform from postdissection fiber length in human skeletal muscle fibers.

Experimental techniques in single human skeletal muscle cells require manual dissection. Unlike other mammalian species, human skeletal muscle is characterized by a heterogeneous mixture of myosin heavy chain (MHC) isoforms, typically used to define "fiber type," which profoundly influences cellular function. Therefore, it is beneficial to predict MHC isoform at the time of dissection, facilitating a more balanced fiber-type distribution from a potentially imbalanced sample. Although researchers performing single fiber dissection report predicting fiber-type based on mechanical properties of fibers upon dissection, a rigorous examination of this approach has not been performed. Therefore, we measured normalized fiber length (expressed as a % of the length of the bundle from which the fiber was dissected) in single fibers immediately following dissection. Six hundred sixty-eight individual fibers were dissected from muscle tissue samples from healthy, young adults to assess whether this characteristic could differentiate fibers containing MHC I ("slow" fiber type) or not ("fast" fiber type). Using receiver operator characteristic (ROC) curves, we found that differences in normalized fiber length (114 ± 13%, MHC I; 124 ± 17%, MHC IIA, P < 0.01) could be used to predict fiber type with excellent reliability (area under the curve = 0.72). We extended these analyses to include older adults (2 females, 1 male) to demonstrate the durability of this approach in fibers with likely different morphology and mechanical characteristics. We report that MHC isoform expression in human skeletal muscle fibers can be predicted at the time of dissection, regardless of origin.NEW & NOTEWORTHY A priori estimation of myosin heavy chain (MHC) isoform in individual muscle fibers may bias the relative abundance of fiber types in subsequent assessment. Until now, no standardized assessment approach has been proposed to characterize fibers at the time of dissection. We demonstrate an approach based on normalized fiber length that may dramatically bias a sample toward slow twitch (MHC I) or fast twitch (not MHC I) fiber populations.

Characterisation of hypertrophic response and the effect of a GATA4-targeting compound on the phenotype of hypertrophic cardiomyopathy patient-derived hiPSC-cardiomyocytes

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.

383 Effect of Feeding Ractopamine to Finisher Lard-Type Mangalica Pigs on Longissimus Muscle Fiber Myosin-Heavy Chain Isoform Expression and Cross-Sectional Area

Abstract Heritage breeds such as Mangalica pigs are gaining popularity in US niche markets and currently sold as high-quality meat due to greater meat quality characteristics. Research has demonstrated that feeding the β-adrenergic agonist ractopamine hydrochloride (RAC), stimulates hypertrophic growth of type-2, fast-twitch glycolytic muscle fibers via mTOR signaling resulting in greater myofibrillar protein synthesis. Therefore, the current study evaluated the effect of feeding RAC to Mangalica pigs during the finisher phase on longissimus muscle fiber myosin heavy-chain (MHC) isoform expression and cross-sectional area. Mangalica pigs were randomly assigned finisher diets for 21 d with 20 ppm (RAC; n = 8) or without (CTL; n = 6). Longissimus muscle samples were collected during carcass fabrication, flash-frozen in liquid nitrogen and stored at -80 °C. Immunofluorescence staining of 5-µm-thick cryosections to identify Type-1 (Developmental Studies Hybridoma Bank (DSHB) antibody Ba-D5), Type-2a (DSHB SC-71), and 2x (DSHB BF-35) MHC positive fibers. Type 2b fibers were determined by subtraction. Fibers in each image (≥ 150) were assessed for fiber type and their cross-sectional area (CSA) at 200X magnification. Data were analyzed as a one-way ANOVA using the GLIMMIX procedure of SAS V9.4. Means were separated with the PDIFF option and declared different when P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.1000. Feeding RAC decreased the density of Type 2a (P = 0.0136) and Type 2b (P = 0.0186) fibers in the longissimus muscle of Mangalica pigs. A tendency for RAC to reduce the density of Type 1, slow-twitch (6.61 vs. 16.14 fiber per mm2; P = 0.0505) fibers and increase the density of primarily oxidative fibers expressing a combination of Type 1 + Type 2a MHC isoforms (10.23 vs. 2.88%; P = 0.0524) was also observed. When analyzed as a proportion of total fibers, RAC reduced the proportion of fibers expressing only Type 2a (11.8 vs. 6.70%; P = 0.0468) and Type 2b (0.19 vs. 1.32%; P = 0.0101) MHC isoforms but increased the percentage of fibers expressing both Type 1 + Type 2a MHC isoforms (4.66 vs. 1.13%; P = 0.0280). Feeding RAC did not impact the density or proportion of fibers expressing Type 2x MHC (P ≥ 0.386). Mean longissimus muscle fiber CSA was not impacted by feeding Mangalica pigs RAC for 21 d (P = 0.4815). The CSA of the smallest and largest fibers nor fiber distribution were influenced by RAC inclusion in the diet of Mangalica pigs (P ≥ 0.484). In conclusion, feeding RAC Mangalica pigs impacted fiber MHC isoform expression in the longissimus muscle without altering fiber CSA. Future studies will explore the impact of RAC on the CSA of the various fiber types as well as the impact on other muscles in the Mangalica pig.

Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the β-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively β-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than β-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/β-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted β-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

Sex differences in skeletal muscle size, function, and myosin heavy chain isoform expression during post-injury regeneration in mice.

Skeletal muscle regeneration is an essential process to restore muscle function after injury and is influenced by various factors. Despite the known importance of sex hormones in muscle regeneration, whether and what sex difference exists in this process is still unclear. In this study, we provide evidence for a clear sex difference in muscle regeneration in mice. At 7 and 14 days after barium chloride-induced muscle injury, female mice showed a faster recovery of muscle fiber size than males. Consistently, muscle force in female mice was restored faster than in males after injury, and this functional difference was maintained at 14 months of age when regenerative capacity declined. Myosin heavy chain isoform profiling and fatigability test revealed dynamic remodeling of myosin heavy chain isoform expression including a type IIB to IIA/X MHC transition and reduced fatigability in regenerated muscles compared to uninjured muscles. A significant sex difference was detected in myosin heavy chain IIX content, although this did not lead to different fatigability. Together, our results suggest that sex is an important determinant of the recovery of regenerating skeletal muscle and is partially involved in the remodeling of myosin heavy chain isoforms during muscle regeneration.

Expression of MyHC-15 and -2x in human muscle spindles: An immunohistochemical study.

To build on the existing data on the pattern of myosin heavy chain (MyHC) isoforms expression in the human muscle spindles, we aimed to verify whether the 'novel' MyHC-15, -2x and -2b isoforms are co-expressed with the other known isoforms in the human intrafusal fibres. Using a set of antibodies, we attempted to demonstrate nine isoforms (15, slow-tonic, 1, α, 2a, 2x, 2b, embryonic, neonatal) in different regions of intrafusal fibres in the biceps brachii and flexor digitorum profundus muscles. The reactivity of some antibodies with the extrafusal fibres was also tested in the masseter and laryngeal cricothyreoid muscles. In both upper limb muscles, the expression of slow-tonic isoform was a reliable marker for differentiating positive bag fibres from negative chain fibres. Generally, bag1 and bag2 fibres were distinguished in isoform 1 expression; the latter consistently expressed this isoform over their entire length. Although isoform 15 was not abundantly expressed in intrafusal fibres, its expression was pronounced in the extracapsular region of bag fibres. Using a 2x isoform-specific antibody, this isoform was demonstrated in the intracapsular regions of some intrafusal fibres, particularly chain fibres. To the best of our knowledge, this study is the first to demonstrate 15 and 2x isoforms in human intrafusal fibres. However, whether the labelling with an antibody specific for rat 2b isoform reflects the expression of this isoform in bag fibres and some extrafusal ones in the specialised cranial muscles requires further evaluation. The revealed pattern of isoform co-expression only partially agrees with the results of previous, more extensive studies. Nevertheless, it may be inferred that MyHC isoform expression in intrafusal fibres varies along their length, across different muscle spindles and muscles. Furthermore, the estimation of expression may also depend on the antibodies utilised, which may also react differently with intrafusal and extrafusal fibres.

Depressed myocardial cross-bridge cycling kinetics in a female guinea pig model of diastolic heart failure.

Cardiac hypertrophy is associated with diastolic heart failure (DHF), a syndrome in which systolic function is preserved but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling are poorly understood. Accordingly, chronic pressure overload was induced by surgically banding the thoracic ascending aorta (AOB) in ∼400 g female Dunkin Hartley guinea pigs (AOB); Sham-operated age-matched animals served as controls. Guinea pigs were chosen to avoid the confounding impacts of altered myosin heavy chain (MHC) isoform expression seen in other small rodent models. In vivo cardiac function was assessed by echocardiography; cardiac hypertrophy was confirmed by morphometric analysis. AOB resulted in left ventricle (LV) hypertrophy and compromised diastolic function with normal systolic function. Biochemical analysis revealed exclusive expression of β-MHC isoform in both sham control and AOB LVs. Myofilament function was assessed in skinned multicellular preparations, skinned single myocyte fragments, and single myofibrils prepared from frozen (liquid N2) LVs. The rates of force-dependent ATP consumption (tension-cost) and force redevelopment (Ktr), as well as myofibril relaxation time (Timelin) were significantly blunted in AOB, indicating reduced cross-bridge cycling kinetics. Maximum Ca2+ activated force development was significantly reduced in AOB myocytes, while no change in myofilament Ca2+ sensitivity was observed. Our results indicate blunted cross-bridge cycle in a β-MHC small animal DHF model. Reduced cross-bridge cycling kinetics may contribute, at least in part, to the development of DHF in larger mammals, including humans.

Tumor Necrosis Factor-α Mediates Inflammation-induced Early-Stage Left Ventricular Systolic Dysfunction.

Elevated systemic inflammation contributes to pathogenesis of heart failure with preserved ejection fraction (HFpEF), but molecular mechanisms are poorly understood. Although left ventricular (LV) diastolic dysfunction is the main cause of HFpEF, subclinical systolic dysfunction also contributes. We have previously shown that rats with collagen-induced arthritis (CIA) have systemic inflammation, LV diastolic dysfunction, and that increased circulating TNF-α contributes to inflammation-induced HFpEF pathogenesis, but does not mediate LV diastolic dysfunction in CIA rats. Contribution of systemic inflammation to dysfunction of the active process of LV diastolic and systolic function are unknown. In the present study, we used the CIA rat model to investigate the effects of systemic inflammation and TNF-α blockade on systolic function, and mRNA expression of genes involved in active diastolic relaxation and of myosin heavy chain (MyHC) isoforms. Collagen inoculation and TNF-α blockade did not affect LV mRNA expression of genes that mediate active LV diastolic function. Collagen-induced inflammation impaired LV global longitudinal strain ( P = 0.03) and velocity ( P = 0.04). This impairment of systolic function was prevented by TNF-α blockade. Collagen inoculation decreased mRNA expression of α-MyHC ( Myh6, P = 0.03) and increased expression of β-MyHC ( Myh7, P = 0.0002), a marker, which is upregulated in failing hearts. TNF-α blockade prevented this MyHC isoform-switch. These results show that increased circulating TNF-α changes the relative expression of MyHC isoforms, favoring β-MyHC, which may underlie changes in contractile function that impair systolic function. Our results indicate that TNF-α initiates early-stage LV systolic, rather than LV diastolic dysfunction.

Expression of neurotransmitters, vasculogenesis markers and myosin heavy chain isoforms in the masseter muscle of senescence-accelerated mouse prone 8 mice.

Learning and memory deficits and pathologic changes in the hippocampus caused by toothlessness and soft diet feeding are related to reduced masseter muscle (MM) function. Myosin heavy chain (MyHC) isoform expression in the MM also changes under different chewing conditions. The neurotransmitter calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor A (VEGF-A) are involved in MM formation. However, the relationship between CGRP, VEGF-A and MyHC isoforms in the MM in the senescence-accelerated mouse prone 8 (SAMP8) strain, a model of learning and memory deficits, remains unclear. Changes in CGRP, VEGF-A, vasculogenesis marker, and MyHC isoform mRNA expression in the MMs of aging SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice was investigated through quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. qRT-PCR revealed obviously high CGRP levels in the SAMP8 mouse MM (p<0.001). MyHC-IId/x mRNA expression in the MM was higher in 24-week-old SAMP8 mice than 24-week-old SAMR1 mice (p<0.001) but lower in slow-MyHC SAMP8 mice than SAMR1 mice (p<0.001). CGRP mRNA was observed on the muscle fibers of the SAMP8 mouse MM but not the SAMR1 mouse MM through in situ hybridization. Principal component analysis (PCA) revealed strong positive contributions of SAMP8-MyHC-IId/x, SAMP8-CGRP, SAMR1-MyHC-emb, SAMR1-CGRP, SAMR1-VEGF-A, SAMR1-CD31, SAMP8-VEGF-A, and SAMP8-CD31 in the MM at 12 and 24 weeks. CGRP is also key for the MyHC-IId/x and slow-MyHC patterns in the MMs of SAMP8 mice.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/β myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81mm/s, NTG: 338.7 ± 28.07mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

Atrial cardiomyocytes from a canine model of atrial fibrillation demonstrate reduced force development and altered expression of contractile proteins.

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and associates with worse outcomes in cardiac contractile-related diseases including cardiomyopathy. As most studies surrounding AF focus on the electrophysiology of the disease, there is little known about cardiomyocyte contractile function in AF. We used atrial tissue from an established atrial tachypacing canine model of AF. Whole protein lysates were collected from the atria to conduct mass spectrometry analysis showing dysregulation of numerous molecular pathways, including contractility. Skinned atrial cardiomyocytes were isolated from the tissue for force calcium and passive tension experiments. Force calcium experiments showed a significant reduction in the maximum force of contraction of AF cardiomyocytes. To explain this, myosin heavy chain (MHC) isoform expression was evaluated because isoform switching is known to alter force of contraction. Decreased percent beta expression was found, explaining the reduction of force due to MHC alpha's capacity for reduced force of contraction. AF cardiomyocytes showed increased calcium sensitivity of force development, which may translate to prolonged contraction with slower relaxation in vivo. A significant reduction in S100A1 was seen in the AF samples via mass spectrometry, which is associated with increased calcium sensitivity. Prolonged contraction along with reduced force of each contraction could support the development of atrial clots. Atrial cardiomyocytes from the AF hearts demonstrated a significant reduction in the passive tension. Evaluation of titin isoform expression, which is known to alter passive tension, showed increased titin degradation, supporting the capacity for atrial dilation in vivo. Discerning the role of contractility in the pathology of atrial fibrillation could provide novel avenues of AF treatment, especially in instances where it is associated with contractile diseases.

Differential expression of myosin heavy chain isoforms type II in skeletal muscles of polar and black bears.

In this study, the pattern of myosin heavy chain (MHC) isoforms expression in skeletal muscles of the trunk, forelimb and hindlimb in Polar Bear (PB) Ursus maritimus; American Black Bear (AmBB), Ursus americanus and Asian Black Bear (AsBB), Ursus thibetanus was analysed by immunohistochemistry and SDS-PAGE. Results showed that slow (MHC-I) and fast (MHC-II) isoforms exist in muscles of bears. Type II fibres were classified further into Type IIa and IIx in PB but not in AsBB and AmBB. The distribution of Type I and Type II fibres in the trunk, forelimb and hindlimb varied based on muscle type and animal species. The proportions of Type I fibres formed approximately one-third of muscle composition in PB (trunk, 32.0%; forelimb, 34.7%; hindlimb, 34.5%) and a half in both AsBB and AmBB whereas Type IIa and IIx formed approximately two-third in PB (trunk, 68.0%; forelimb, 65.3%; hindlimb, 65.5%) and a half of Type II in both AmBB and AsBB. PB is a good swimmer, lives in Arctic Ocean on slippery ice catching aquatic mammals such as seals and is larger in size compared to the medium sized AmBB (living in forest) and AsBB (arboreal). The results suggest that in bears, there is greater diversity in MHC isoforms II, being expressed in selected fast contracting skeletal muscles in response to variety of environments, weight bearing and locomotion.

Anatomical and molecular analyses of the deltoid muscle in chimpanzees (Pan troglodytes) and modern humans (Homo sapiens): Similarities and differences due to the uses of the upper extremity.

In the deltoid muscles of Pan troglodytes and Homo sapiens, we have analyzed the muscle architecture and the expression of the myosin heavy chain (MHC) isoforms. Our aim was to identify differences between the two species that could be related to their different uses of the upper limb. The deltoid muscle of six adult Pan troglodytes and six adult Homo sapiens were dissected. The muscle fascicle length (MFL) and the physiological cross-sectional area (PCSA) of each muscle were calculated in absolute and normalized values. The expression pattern of the MHC-I, MHC-IIa and MHC-IIx isoforms was analyzed in the same muscles by real-time polymerase chain reaction. Only the acromial deltoid (AD) presented significant architectural differences between the two species, with higher MFL values in humans and higher PCSA values in chimpanzees. No significant differences in the expression pattern of the MHC isoforms were identified. The higher PCSA values in the AD of Pan troglodytes indicate a greater capacity of force generation in chimpanzees than in humans, which may be related to a greater use of the upper limb in locomotion, specifically in arboreal locomotion like vertical climbing. The functional differences between chimpanzees and humans in the deltoid muscle are more related to muscle architecture than to a differential expression of MHC isoforms.

Postnatal Phrenic Motor Neuron and Diaphragm Maturation in the Mouse

In rats, there is a strong association between the expansion of phrenic motor neuron (PhMN) size (from ~21‐28 days postnatal), the emergence of mature diaphragm muscle (DIAm) myosin heavy chain (MyHC2X and MyHC2B) isoform expression, alongside the manifestation of mature morphological and functional properties. Despite mice models being de rigueur for developmental investigations, the postnatal development of mouse PhMN and DIAm properties have not been comprehensively characterized. Using wildtype C57 mice at ages P7, P14, P21, P28, and &gt;3 months (maturity) we evaluated PhMN and DIAm physiologic properties. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging and PhMN assessment. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh‐frozen, with 8 µm cross‐sections immuno‐labelled for MyHCslow, MyHC2A, MyHC2X and MyHC2X and processed for fluorescent assessment. The number of PhMNs remained stable throughout postnatal life (~195), while PhMN surface area increased steadily, ~10% from P14 to P21, P21 to P28, and P28 to maturity, with overall ~30% increase from P14 to maturity. The biggest changes in number of primary dendrites occurred between P14 and P28 and between P28 and maturity, 25% decrease in primary dendrites between each group. DIAm maximum Po increased in two phases, 3.5‐fold from P7 to P14 and remained similar until P21. The second large increase was ~3‐fold from P21 to P28 and remained steady to maturity. The fatiguability of the DIAm increased with age in two phases, with fatigability being similar from P7 and P14 and increasing by ~20% at P21. Following, fatigability of the DIAm was similar from P21 to P28 and increased by ~35% at maturity. These major PhMN and DIAm changes coincide with the steady growth of PhMN and the emergence of MyHC2X and MyHC2B expression in DIAm from P14 to P21 onwards. The maturation of orderly PhMN recruitment likely depends on an interplay between PhMN size properties and network inputs to a refined dendritic tree. We show that DIAm muscle fiber maturation in mice hinges on the expression of MyHC2X and MyHC2B isoforms that is associated with an ~10 fold increase in DIAm force‐generation capacity.

HDAC4 Is Indispensable for Reduced Slow Myosin Expression at the Early Stage of Hindlimb Unloading in Rat Soleus Muscle.

It is well known that reduced contractile activity of the main postural soleus muscle during long-term bedrest, immobilization, hindlimb unloading, and space flight leads to increased expression of fast isoforms and decreased expression of the slow isoform of myosin heavy chain (MyHC). The signaling cascade such as HDAC4/MEF2-D pathway is well-known to take part in regulating MyHC I gene expression. Earlier, we found a significant increase of HDAC4 in myonuclei due to AMPK dephosphorylation during 24 h of hindlimb unloading via hindlimb suspension (HU) and it had a significant impact on the expression of MyHC isoforms in rat soleus causing a decrease in MyHC I(β) pre-mRNA and mRNA expression as well as MyHC IIa mRNA expression. We hypothesized that dephosphorylated HDAC4 translocates into the nuclei and can lead to a reduced expression of slow MyHC. To test this hypothesis, Wistar rats were treated with HDAC4 inhibitor (Tasquinimod) for 7 days before HU as well as during 24 h of HU. We discovered that Tasquinimod treatment prevented a decrease in pre-mRNA expression of MyHC I. Furthermore, 24 h of hindlimb suspension resulted in HDAC4 nuclear accumulation of rat soleus but Tasquinimod pretreatment prevented this accumulation. The results of the study indicate that HDAC4 after 24 h of HU had a significant impact on the precursor MyHC I mRNA expression in rat soleus.

Skeletal muscle cellular contractile dysfunction after anterior cruciate ligament reconstruction contributes to quadriceps weakness at 6-month follow-up.

Muscle dysfunction following anterior cruciate ligament reconstruction (ACLR) may evolve from alterations in muscle contractility at the myofilament protein level. Using a prospective, within-subject case-control design, we evaluated cellular-level contractility, cross-sectional area (CSA), and myosin heavy chain (MHC) isoform expression on single muscle fibers 3 weeks post ACLR, and evaluated their relationship to whole muscle strength and patient-oriented outcomes 6 months post operation. Biopsies of the vastus lateralis were performed 3 weeks post ACLR in 11 subjects (5 females, mean age ± SD = 24.7 ± 6.5 years, height = 172.7 ± 8.2 cm, mass = 75.7 ± 12.5 kg) following first-time ACL rupture and whole muscle strength and self-reported pain, function, and quality of life assessed 6 months post ACLR. At 3 weeks post ACLR, force production was reduced (p < 0.01) in MHC I (-36%) and IIA (-48%) fibers compared with the non-injured leg. When force production was expressed relative to CSA to account for fiber atrophy, reductions remained in MHC IIA fibers (-40%; p < 0.001), but MHC I fibers showed only a trend toward being lower (-13%; p = 0.09). Finally, skeletal muscle fiber functional deficits at 3 weeks post ACLR were associated with whole muscle weakness and less favorable patient-reported outcomes at 6-month follow-up. Thus, ACLR promotes early cellular contractile dysfunction that may contribute to decreased whole muscle strength and patient function, and increased patient-reported symptoms, at 6-month follow-up.

Skeletal muscle and fiber type-specific intramyocellular lipid accumulation in obese mice.

ABSTRACTIn obesity, accumulation of lipid droplets in skeletal muscle fibers and a shift towards fast muscle fiber types can both contribute to insulin resistance. However, it is not yet clear how intramyocellular lipid accumulation and fiber type changes are associated. Therefore, we investigated to what extent the lipids accumulated in a fiber type-specific manner in the functionally similar fast-, intermediate- and slow-twitch gastrocnemius, plantaris, and soleus muscles, respectively, in high-fat diet-induced obese 54-week-old female C57BL/6JOlaHsd mice (n = 9) compared to control standard-diet-treated lean mice (n = 9). A high-fat diet was administered for 26 weeks. Fiber-type specific intramyocellular lipid content analysis and muscle fiber typing were performed using histochemical analysis of lipids with Sudan Black and immunohistochemical analysis of myosin heavy chains on serial sections of skeletal muscles. Compared to the lean mice, the lipid accumulation was most prominent in types 2a and 2x/d fibers (p < 0.05) of fast-twitch gastrocnemius and intermediate plantaris muscles in the obese mice, while in slow-twitch soleus muscle, there was no significant lipid accumulation in the obese animals. Furthermore, the slow-twitch soleus muscle of the obese mice with no significant change in muscle fiber diameters exhibited the most pronounced shift towards fast-type myosin heavy chain isoform expression (p < 0.05). In contrast, the fast-twitch and intermediate-twitch gastrocnemius and plantaris muscles, respectively, in which the muscle fiber diameters increased (p < 0.05), were more resistant toward myosin heavy chain expression changes. In conclusion, we demonstrated both muscle- and fiber-type specificity in intramyocellular lipid accumulation in obese mice, suggesting that in obesity, similar muscle fiber types in different muscles accumulate lipids differentially.