Myosin Heavy Chain Gene Expression Research Articles

Abstract P1164: Elevated Erk1/2 Signaling Contributes To The Development Of Diabetic Cardiomyopathy

Background: About 19%-26% of the obese patients develop diabetic cardiomyopathy (DCM) characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Although many studies have investigated the potential causes of DCM, the pathophysiology of this disease has yet been to be fully characterized. Objective: To determine whether extracellular signal-regulated kinase 1/2 (ERK1/2) are involved in the development of DCM. Materials and Methods: Both streptozotocin (STZ)-induced type I and db/db type II diabetic mice were utilized to compare the ERK1/2 phosphorylation level in mouse hearts. To determine the gain-of-function of ERK1/2 signaling in DCM, both control and dual specific phosphatase 6 and 8 knockout mice (DKO) mice were administered streptozotocin (STZ) to induce elevated serum glucose. After 12 weeks of treatment, mouse body weight, heart weight/histology, and disease markers were compared. As a loss-of-function study, U0126 was utilized to inhibit ERK1/2 activity in both streptozotocin (STZ)-induced type I and db/db type II diabetic mice to determine whether DCM markers are influenced. Results: In both type I and II diabetic mouse hearts, ERK1/2 phosphorylation level was significantly elevated. STZ treatment for 2 weeks induced similar increases in serum glucose in both WT and DKO mice. After 12 weeks of STZ treatment, DKO mice developed greater cardiac atrophy and increased levels of ANF, BNP, beta myosin heavy chain gene expression. In comparison, U0126-mediated pharmacological inhibition of ERK1/2 for 12 weeks significantly improved the cardiac function in both type I and II diabetic mouse models. Conclusion: Elevated ERK1/2 signaling contributes to the process of DCM, suggesting ERK1/2 could be therapeutic targets for the treatment of this disease.

Different myocardial microRNA expression patterns are observed in pressure overload- and volume overload-induced chronic heart failure

Abstract Introduction MicroRNAs (miRNA) are short, single-stranded non-coding RNA molecules that regulate gene expression on the post-transcriptional level. Dysregulation of distinct miRNAs has been found to contribute to the development of chronic heart failure (CHF). However, whether distinct types of CHF are associated with different miRNA expression patterns is debated. Aim To characterize left ventricular (LV) miRNA expression in rat models of pressure overload (PO) versus volume overload (VO)-induced CHF. Methods Transverse aortic constriction (TAC) was performed to evoke PO-induced CHF. Aortocaval fistula (ACF) was created to establish VO-induced CHF. Age-matched sham-operated rats served as controls for TAC (ShamT) and ACF (ShamA), respectively. Pressure-volume analysis, echocardiography, histology and quantitative real-time PCR were carried out to assess alterations of the LV. Global miRNA expression profiling was performed using Nanostring technology. Bioinformatics analysis of differentially expressed miRNAs was also carried out to predict relevant miRNA-target interactions. Results Reduced LV systolic function (ejection fraction: 38±5 vs. 65±2% TAC vs. ShamT, 55±3 vs. 67±3%, ACF vs. ShamA, P<0.01) as well as elevated myocardial B-type natriuretic peptide and increased β-to-α myosin heavy chain gene expression confirmed the development of pathological remodeling and CHF in both models. Nevertheless, characteristic differences could be observed in LV morphology and ultrastructure. Accordingly, the TAC model was associated with robustly increased wall thickness, concentric LV hypertrophy and marked fibrotic remodeling. On the contrary, LV dilatation, eccentric LV hypertrophy and moderate fibrosis were the main morphometric findings in the ACF model. A group of miRNA (rno-miR-130a, 132, 199a-5p, 21, 210, 27b, 326) showed similar alterations in both phenotypes of CHF. However, other miRNAs demonstrated unique (specific to TAC: rno-miR-148b-3p, 150, 199a-3p, 203, 23b, 27b, let-7e; specific to ACF: rno-miR-140, 142-3p, 17-5p, 195, 20a, 204, 214, 27a, 29b, 322, 365, 425, 450a, let-7i) LV expressional changes in distinct phenotypes of CHF. In silico bioinformatics analysis revealed that the altered miRNA expression pattern predominantly controls the cardiac neural crest cell development, the inositol-phosphate pathway and the expression of microtubules-binding proteins. In contrast, alterations in the expression of genes responsible for redox state and epithelial-mesenchymal transition were modified only in the ACF group. Despite of the different signaling cascades, expression of Arhgap12 (Rho GTPase activating protein 12) was predicted to be strongly inhibited in both CHF models. Conclusions PO and VO-induced CHF are associated with unique miRNA expression patterns, which drive different signaling pathways. miRNA-controlled downregulation of Arhgap12 might represent a common feature in both phenotypes of CHF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ÚNKP-20-4-II-SE-20/New National Excellence Program of the Ministry of Human CapacitiesNVKP_16-1–2016-0017 (“National Heart Program”) National Research, Development and Innovation Fund of Hungary

Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth.

Feeding ractopamine (RAC), a β-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P < 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P < 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P < 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, β 1-adrenergic receptor (AR), and β 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P < 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P < 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P < 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. β 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the β 2-AR gene was decreased (P < 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and β 2-AR gene expression in porcine skeletal muscles independent of growth.

Thermal acclimation of rainbow trout myotomal muscle, can trout acclimate to a warming environment?

Climate change is a looming threat to the planet. Cold-water aquatic species will face significant physiological challenges due to elevated summer temperatures. Salmonids, such as rainbow trout (Oncorhynchus mykiss) maintain fidelity to native streams, limiting their ability to mitigate the impact of climate change through migration. We examined how rainbow trout swimming performance and muscle function were shaped by the thermal environment. We hypothesized that trout would show slower muscle contractile properties and slower swimming performance with long-term exposure to warmer water. For fish held at either 10 °C or 20 °C, maximum steady swimming speed (Ucrit) was determined, and contractile properties of both fast-twitch (white) and slow-twitch (red) myotomal muscle were examined. In addition, immunohistochemistry and quantitative PCR were used to assess changes in myosin content of the myotomal muscle in response to holding temperature. Rainbow trout exposed to warm water for six weeks displayed relatively limited thermal acclimation response. When tested at a common temperature (10 °C), 20 °C acclimated fish had modestly slower muscle performance compared to 10 °C acclimated fish. Significant differences in swimming performance and muscle contractile properties were primarily at colder test temperatures (e.g. 2 °C for muscle mechanics). Shifts in myosin heavy chain protein composition and myosin heavy chain gene expression in the swimming muscle were observed in white but not red muscle. Our results suggest that rainbow trout will have a limited ability to mitigate elevated environmental temperature through thermal acclimation of their myotomal or swimming muscle.

Regulation of myosin heavy chain antisense long noncoding RNA in human vastus lateralis in response to exercise training

Alterations to muscle activity or loading state can induce changes in expression of myosin heavy chain (MHC). For example, sedentary individuals that initiate exercise training can induce a pronounced shift from IIx to IIa MHC. We sought to examine the regulatory response of MHC RNA in human subjects in response to exercise training. In particular, we examined how natural antisense RNA transcripts (NATs) are regulated throughout the MHC gene locus that includes MYH2 (IIa), MYH1 (IIx), MYH4 (IIb), and MYH8 (Neonatal) in vastus lateralis before and after a 5-wk training regime that consisted of a combination of aerobic and resistance types of exercise. The exercise program induced a IIx to IIa MHC shift that was associated with a corresponding increase in transcription on the antisense strand of the IIx MHC gene and a decrease in antisense transcription of the IIa MHC gene, suggesting an inhibitory mechanism mediated by NATs. We also report that the absence of expression of IIb MHC in human limb muscle is associated with the abundant expression of antisense transcript overlapping the IIb MHC coding gene, which is the opposite expression pattern as compared with that previously observed in rats. The NAT provides a possible regulatory mechanism for the suppressed expression of IIb MHC in humans. These data indicate that NATs may play a regulatory role with regard to the coordinated shifts in MHC gene expression that occur in human muscle in response to exercise training.

Introduction of Specific 3D Micromorphologies in Collagen Scaffolds Using Odd and Even Dicarboxylic Acids.

The construction of scaffolds and subsequent incorporation of cells and biologics have been widely investigated to regenerate damaged tissues. Scaffolds act as a template to guide tissue formation, and their characteristics have a considerable impact on the regenerative process. Whereas many technologies exist to induce specific two-dimensional (2D) morphologies into biomaterials, the introduction of three-dimensional (3D) micromorphologies into individual pore walls of scaffolds produced from biological molecules such as collagen poses a challenge. We here report the use of dicarboxylic acids to induce specific micromorphologies in collagen scaffolds and evaluate their effect on cellular migration and differentiation. Insoluble type I collagen fibrils were suspended in monocarboxylic and dicarboxylic acids of different concentrations, and unidirectional and random pore scaffolds were constructed by freezing and lyophilization. The application of various acids and concentrations resulted in variations in 3D micromorphologies, including wall structure, wall thickness, and pore size. The use of dicarboxylic acids resulted in acid-specific micromorphologies, whereas monocarboxylic acids did not. Dicarboxylic acids with an odd or even number of C-atoms resulted in frayed/fibrillar or smooth wall structures, respectively, with varying appearances. The formation of micromorphologies was concentration-dependent. In vitro analysis indicated the cytocompatibility of scaffolds, and micromorphology-related cell behavior was indicated by enhanced myosin staining and myosin heavy chain gene expression for C2C12 myoblasts cultured on scaffolds with frayedlike micromorphologies compared to those with smooth micromorphologies. In conclusion, porous collagen scaffolds with various intrawall 3D micromorphologies can be constructed by application of dicarboxylic acids, superimposing the second level of morphology to the overall scaffold structure. Acid crystal formation is key to the specific micromorphologies observed and can be explained by the odd/even theory for dicarboxylic acids. Scaffolds with a 3D micrometer-defined topography may be used as a screening platform to select optimal substrates for the regeneration of specific tissues.

MiR-29a activates expression of α-cardiac myosin heavy chain via β1 thyroid hormone receptor in neonatal rats.

MicroRNAs (miRs) are small noncoding RNAs which are regulators of gene expression and also regulate the genes in heart tissues. The aim of the study was to evaluate the effect of miRs on the expression level of myosin heavy chain (MHC), which is responsible for regulation of cardiac functions in neonatal rat ventricular myocytes and mice. The miRs were suppressed in neonatal rat ventricular myocytes using small interfering RNAs (siRNAs) against Dicer followed by evaluation of MHC levels. For in vivo study the C57 black/6 Jacksonian mice were subjected to the transverse aortic constriction (TAC) procedure. The Dicer siRNA suppressed the endogenous miRs and the α-MHC gene but failed to down-regulate the β-MHC. Among the 17 selected miRs, miR-29a was found to up-regulate the α-MHC gene significantly but not β-MHC. The expression of α-MHC was suppressed by silencing the expression of miR-29a. Bioinformatics study done by TargetScan suggested thyroid hormone receptor-β1 (TR-β1) as a potential target of miR-29a. Additionally, miR-29a was found to regulate the expression of α-MHC via TR-β1 signaling. The findings of the present study indicated that miR-29a modulates expression of α-the MHC gene by targeting TR-β1 in cardiac cells. The study may provide a new direction for treating cardiac failure and cardiac hypertrophy.

Combination of laser and human adipose-derived stem cells in repair of rabbit anal sphincter injury: a new therapeutic approach

BackgroundAnal sphincter injury leads to fecal incontinence. Based on the regenerative capability of laser and human adipose-derived stem cells (hADSCs), this study was designed to assess the effects of co-application of these therapies on anal sphincter recovery after injury.DesignMale rabbits were assigned to equal groups (n = 7) including control, sphincterotomy, sphincterotomy treated with laser (660 nm, 90 s, immediately after sphincterotomy, daily, 14 days), hADSCs (2 × 106 hADSCs injected into injured area of the sphincter immediately after sphincterotomy), and laser + hADSCs. Ninety days after sphincterotomy, manometry and electromyography were performed, sphincter collagen content was evaluated, and Ki67, myosin heavy chain (MHC), skeletal muscle alpha-actin (ACTA1), vascular endothelial growth factor A (VEGFA), and vimentin mRNA gene expression were assessed.ResultsThe laser + hADSCs group had a higher resting pressure compared with the sphincterotomy (p < 0.0001), laser (p < 0.0001), and hADSCs (p = 0.04) groups. Maximum squeeze pressure was improved in all treated animals compared with the sphincterotomized animals (p < 0.0001), without a significant difference between treatments (p > 0.05). In the laser + hADSCs group, motor unit numbers were higher than those in the laser group (p < 0.0001) but did not differ from the hADSCs group (p = 0.075). Sphincterotomy increased collagen content, but the muscle content (p = 0.36) and collagen content (p = 0.37) were not significantly different between the laser + hADSCs and control groups. Laser + hADSCs increased ACTA1 (p = 0.001) and MHC (p < 0.0001) gene expression compared with laser or hADSCs alone and was associated with increased VEGFA (p = 0.009) and Ki67 mRNA expression (p = 0.01) and decreased vimentin mRNA expression (p < 0.0001) compared with laser.ConclusionThe combination of laser and hADSCs appears more effective than either treatment alone for promoting myogenesis, angiogenesis, and functional recovery after anal sphincterotomy.

Differentiation of Bioengineered Skeletal Muscle within a 3D Printed Perfusion Bioreactor Reduces Atrophic and Inflammatory Gene Expression.

Bioengineered skeletal muscle tissues benefit from dynamic culture environments which facilitate the appropriate provision of nutrients and removal of cellular waste products. Biologically compatible perfusion systems hold the potential to enhance the physiological biomimicry of in vitro tissues via dynamic culture, in addition to providing technological advances in analytical testing and live cellular imaging for analysis of cellular development. To meet such diverse requirements, perfusion systems require the capacity and adaptability to incorporate multiple cell laden constructs of both monolayer and bioengineered tissues. This work reports perfusion systems produced using additive manufacturing technology for the in situ phenotypic development of myogenic precursor cells in monolayer and bioengineered tissue. Biocompatibility of systems 3D printed using stereolithography (SL), laser sintering (LS), and PolyJet outlined preferential morphological development within both SL and LS devices. When exposed to intermittent perfusion in the monolayer, delayed yet physiologically representative cellular proliferation, MyoD and myogenin transcription of C2C12 cells was evident. Long-term (8 days) intermittent perfusion of monolayer cultures outlined viable morphological and genetic in situ differentiation for the live cellular imaging of myogenic development. Continuous perfusion cultures (13 days) of bioengineered skeletal muscle tissues outlined in situ myogenic differentiation, forming mature multinucleated myotubes. Here, reductions in IL-1β and TNF-α inflammatory cytokines, myostatin, and MuRF-1 atrophic mRNA expression were observed. Comparable myosin heavy chain (MyHC) isoform transcription profiles were evident between conditions; however, total mRNA expression was reduced in perfusion conditions. Decreased transcription of MuRF1 and subsequent reduced ubiquitination of the MyHC protein allude to a decreased requirement for transcription of MyHC isoform transcripts. Together, these data appear to indicate that 3D printed perfusion systems elicit enhanced stability of the culture environment, resulting in a reduced basal requirement for MyHC gene expression within bioengineered skeletal muscle tissue.

Increase in muscle power is associated with myofibrillar ATPase adaptations during resistance training.

What is the central question of this study? The aim of this study was to examine the effects of resistance training on gains in the external mechanical power output developed during climbing and myofibrillar ATPase activity in rats. What is the main finding and its importance? Using rapid flow quench experiments, we show that resistance training increases both the power output and the myofibrillar ATPase activity in the flexor digitorum profundus, biceps and deltoid muscles. Data fitting reveals that these functional ameliorations are explained by an increase in the rate constant of liberation of ATP hydrolysis products and contribute to performance gains. Skeletal muscle shows a remarkable plasticity that permits functional adaptations in response to different stimulations. To date, modifications of the proportions of myosin heavy chain (MHC) isoforms and increases in fibre size are considered to be the main factors providing sarcomeric plasticity in response to exercise training. In this study, we investigated the effects of a resistance training protocol on the myofibrillar ATPase (m-ATPase) cycle, muscle performance (power output) and MHC gene expression. For this purpose, 8-week-old Wistar Han rats were subjected to 4weeks of resistance training, with five sessions per week. Muscle samples of flexor digitorum profundus (FDP), biceps and deltoid were collected and subjected to RT-qPCR analyses and assessment of m-ATPase activity with rapid flow quench apparatus. Training led to a significant increase in muscle mass, except for the biceps, and in total mechanical power output (+135.7%, P<0.001). A shift towards an intermediate fibre type (i.e. MHC2x-to-MHC2a isoform transition) was also observed in biceps and FDP but not in the deltoid muscle. Importantly, rapid flow quench experiments revealed an enhancement of the m-ATPase activity during contraction at maximal velocity (kF ) in the three muscles, with a more marked effect in FDP (+242%, P<0.001). Data fitting revealed that the rate constant of liberation of ATP hydrolysis products (k3 ) appears to be the main factor influencing the increase in m-ATPase activity. In conclusion, the data showed that, in addition to classically observed changes in MHC isoform content and fibre hypertrophy, m-ATPase activity is enhanced during resistance training and might contribute significantly to performance gains.

AMP-Activated Protein Kinase as a Key Trigger for the Disuse-Induced Skeletal Muscle Remodeling

Molecular mechanisms that trigger disuse-induced postural muscle atrophy as well as myosin phenotype transformations are poorly studied. This review will summarize the impact of 5′ adenosine monophosphate -activated protein kinase (AMPK) activity on mammalian target of rapamycin complex 1 (mTORC1)-signaling, nuclear-cytoplasmic traffic of class IIa histone deacetylases (HDAC), and myosin heavy chain gene expression in mammalian postural muscles (mainly, soleus muscle) under disuse conditions, i.e., withdrawal of weight-bearing from ankle extensors. Based on the current literature and the authors’ own experimental data, the present review points out that AMPK plays a key role in the regulation of signaling pathways that determine metabolic, structural, and functional alternations in skeletal muscle fibers under disuse.

Excessive training induces molecular signs of pathologic cardiac hypertrophy.

Chronic exercise induces cardiac remodeling that promotes left ventricular hypertrophy and cardiac functional improvement, which are mediated by the mammalian or the mechanistic target of rapamycin (mTOR) as well as by the androgen and glucocorticoid receptors (GRs). However, pathological conditions (i.e., chronic heart failure, hypertension, and aortic stenosis, etc.) also induce cardiac hypertrophy, but with detrimental function, high levels of proinflammatory cytokines and myostatin, elevated fibrosis, reduced adenosine monophosphate-activated protein kinase (AMPK) activation, and fetal gene reactivation. Furthermore, recent studies have evidenced that excessive training induced an inflammatory status in the serum, muscle, hypothalamus, and liver, suggesting a pathological condition that could also be detrimental to cardiac tissue. Here, we verified the effects of three running overtraining (OT) models on the molecular parameters related to physiological and pathological cardiac hypertrophy. C57BL/6 mice performed three different OT protocols and were evaluated for molecular parameters related to physiological and pathological cardiac hypertrophy, including immunoblotting, reverse transcription polymerase chain reaction, histology, and immunohistochemistry analyses. In summary, the three OT protocols induced left ventricle (LV) hypertrophy with signs of cardiac fibrosis and negative morphological adaptations. These maladaptations were accompanied by reductions in AMPKalpha (Thr172) phosphorylation, androgen receptor, and GR expressions, as well as by an increase in interleukin-6 expression. Specifically, the downhill running-based OT model reduced the content of some proteins related to the mTOR signaling pathway and upregulated the β-isoform of myosin heavy-chain gene expression, presenting signs of LV pathological hypertrophy development.

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.

Effects of increased myocardial tissue concentration of myristic, palmitic and palmitoleic acids on the course of cardiac atrophy of the failing heart unloaded by heterotopic transplantation.

The present experiments were performed to evaluate if increased heart tissue concentration of fatty acids, specifically myristic, palmitic and palmitoleic acids that are believed to promote physiological heart growth, can attenuate the progression of unloading-induced cardiac atrophy in rats with healthy and failing hearts. Heterotopic abdominal heart transplantation (HT(x)) was used as a model for heart unloading. Cardiac atrophy was assessed from the ratio of the native- to-transplanted heart weight (HW). The degree of cardiac atrophy after HT(x) was determined on days 7, 14, 21 and 28 after HT(x) in recipients of either healthy or failing hearts. HT(x) of healthy hearts resulted in 23+/-3, 46+/-3, 48+/-4 and 46+/-4 % HW loss at the four time-points. HT(x) of the failing heart resulted in even greater HW losses, of 46+/-4, 58+/-3, 66+/-2 and 68+/-4 %, respectively (P<0.05). Activation of "fetal gene cardiac program" (e.g. beta myosin heavy chain gene expression) and "genes reflecting cardiac remodeling" (e.g. atrial natriuretic peptide gene expression) after HT(x) was greater in failing than in healthy hearts (P<0.05 each time). Exposure to isocaloric high sugar diet caused significant increases in fatty acid concentrations in healthy and in failing hearts. However, these increases were not associated with any change in the course of cardiac atrophy, similarly in healthy and post-HT(x) failing hearts. We conclude that increasing heart tissue concentrations of the fatty acids allegedly involved in heart growth does not attenuate the unloading-induced cardiac atrophy.

Effect of prior application with and without post-injury treatment with low-level laser on the modulation of key proteins in the muscle repair process.

The aim of the present study was to evaluate the effects of LLLT prior to muscle injury with and without post-injury irradiation on the expression of isoforms of myosin heavy chain (MyHC), calcineurin (CaN), and myostatin during the repair process. Wistar rats were divided into five groups: control (n = 7); injury (n = 21); LLLT + injury (n = 21); injury + LLLT (n = 21), and LLLT + injury + LLLT (n = 21). Cryoinjury was performed on the tibialis anterior (TA) muscle. The injured groups were euthanized at 3, 7, and 14days after injury. LLLT was performed using an infrared laser (780nm) with the following parameters: 10J/cm2, 40mW, 10s per point, 8 points, and 3.2J of total energy. At the end of each period, the TA muscle was removed for the analysis of MyHC, CaN, and myostatin gene expression using real-time PCR. The data were tested statistically by Kruskal-Wallis with Dunn's post hoc test (p < 0.05). The results demonstrated that prior irradiation reduced the mRNA expression of all proteins at 3days. Post irradiation reduced the mRNA expression of MyHC-1, MyHC-2a, MyHC-2b, and CaN at 7days. Prior irradiation combined with post-injury irradiation reduced the mRNA expression of MyHC-2x and CaN at 14days and increased the mRNA expression of myostatin in the same period. In conclusion, different protocols of photobiomodulation can modulate the expression of the different isoforms of MyHC, CaN, and myostatin during the repair process. It is noteworthy that the combination of the prior and post-injury irradiation was the protocol that most promoted changes in the final phase of the repair process.

Role of the cofilin 2 gene in regulating the myosin heavy chain genes in mouse myoblast C2C12 cells.

The cofilin2 (CFL2) and myosin heavy chain(MyHC) genes play a key role in muscle development and myofibrillar formation. The aim of the present study was to investigate the effect of CFL2 on genes involved in fiber formation and the mechanisms underlying this process. Undifferentiated and differentiated C2C12 cells (UDT and DT, respectively) were transfected with CFL2 small interfering RNA (siRNA). CFL2 mRNA and protein levels were assessed using reverse transcription polymerase chain reaction (RT-PCR) and western blotting, respectively. MyHC gene expression in UDT and signaling pathway-related factors were observed with quantitative PCR (RT‑qPCR) and western blotting. Fluorescence microscopy was used to analyze the cytoskeletal effects of CFL2. The mRNA and protein expressions of CFL2, four MyHC isoforms (MyHC-I, MyHC-IIa, MyHC-IIb and MyHC-IIx), p38 mitogen-activated protein kinase, cAMP-response element-binding protein, AMP-activated protein kinaseα1, and myocyte enhancer factor2C,were significantly decreased in UDT. However, extracellular signal-regulated kinase2expression was significantly increased. Slightly decreased CFL2 protein and mRNA expression was observed in DT C2C12 cells transfected with CFL2 siRNA. Fluorescence microscopy revealed a significant decrease of CFL2 in the cytoplasm, but not the nucleus, of UDT, compared with normal cells. These results indicated that the mouse CFL2 gene may be involved in the regulation of MyHC via the key signaling molecules of CFL2-related signaling pathways.

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes.

ObjectiveThis study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula’s extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes.MethodsIn a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction.ResultsThe final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber I, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by 4 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/mL in a TCMF petroleum ether extraction (p<0.05).ConclusionThese results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

Ontogeny of myosin isoform expression and prehensile function in the tail of the gray short-tailed opossum (Monodelphis domestica).

Terrestrial opossums use their semiprehensile tail for grasping nesting materials as opposed to arboreal maneuvering. We relate the development of this adaptive behavior with ontogenetic changes in myosin heavy chain (MHC) isoform expression from 21 days to adulthood. Monodelphis domestica is expected to demonstrate a progressive ability to flex the distal tail up to age 7 mo, when it should exhibit routine nest construction. We hypothesize that juvenile stages (3-7 mo) will be characterized by retention of the neonatal isoform (MHC-Neo), along with predominant expression of fast MHC-2X and -2B, which will transition into greater MHC-1β and -2A isoform content as development progresses. This hypothesis was tested using Q-PCR to quantify and compare gene expression of each isoform with its protein content determined by gel electrophoresis and densitometry. These data were correlated with nesting activity in an age-matched sample of each age group studied. Shifts in regulation of MHC gene transcripts matched well with isoform expression. Notably, mRNA for MHC-Neo and -2B decrease, resulting in little-to-no isoform translation after age 7 mo, whereas mRNA for MHC-1β and -2A increase, and this corresponds with subtle increases in content for these isoforms into late adulthood. Despite the tail remaining intrinsically fast-contracting, a critical growth period for isoform transition is observed between 7 and 13 mo, correlating primarily with use of the tail during nesting activities. Functional transitions in MHC isoforms and fiber type properties may be associated with muscle "tuning" repetitive nest remodeling tasks requiring sustained contractions of the caudal flexors.NEW & NOTEWORTHY Little is understood about skeletal muscle development as it pertains to tail prehensility in mammals. This study uses an integrative approach of relating both MHC gene and protein expression with behavioral and morphometric changes to reveal a predominant fast MHC expression with subtle isoform transitions in caudal muscle across ontogeny. The functional shifts observed are most notably correlated with increased tail grasping for nesting activities.

MiR-30e is negatively regulated by myostatin in skeletal muscle and is functionally related to fiber-type composition.

Myostatin (MSTN) negatively regulates skeletal myogenesis in which microRNAs (miRNAs) also play critical roles. Using miRNA microarrays of skeletal muscle from MSTN-knockout (MSTN-/-) mice, we recently showed that miR-431 is regulated by MSTN signaling. To identify additional miRNAs regulated by MSTN, we re-analyzed these miRNA arrays and validated their expression by quantitative RT-PCR. Herein, we demonstrated that miR-30e was significantly upregulated in skeletal muscle of MSTN-/- mice compared with that of the wild-type littermates. Importantly, the predicted targets of miR-30e are functionally involved in myocyte differentiation and fiber-type formation. Using luciferase reporter gene assays, we further showed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Pgc1α), is a direct target of miR-30e. Overexpression of miR-30e in C2C12 cells significantly decreased Pgc1α and increased type II form of myosin heavy chain gene expression, suggesting that miR-30e functionally associates with glycolytic myofiber formation. Thus, our data indicate that the altered fiber-type composition in MSTN-/- mice are attributable in part to deregulated expression of miR-30e.

Akirin2 promotes slow myosin heavy chain expression by CaN/NFATc1 signaling in porcine skeletal muscle satellite cells.

The objective of this study was to evaluate the effect of Akirin2 on slow myosin heavy chain (slow MyHC, MyHC I) gene expression and its molecular mechanisms. In this study, we showed that the protein expression of Akirin2 in pig slow oxidative Psoas major muscle is higher than that in fast glycolytic tibialis anterior muscle, suggesting that Akirin2 may play a role in myofiber typing. Knockdown of Akirin2 decreased the MyHC I expression and the calcineurin (CaN) activity, and also decreased the expressions of NFATc1 and MCIP1.4. Conversely, overexpression of Akirin2 got the opposite results. Furthermore, inhibition of CaN or knockdown of NFATc1 attenuated Akirin2 overexpression-induced upregulation of MyHC I. Together, these results demonstrate that Akirin2 promotes MyHC I expression via CaN/NFATc1 signaling pathway in porcine skeletal muscle satellite cells.