circSamd4 represses myogenic transcriptional activity of PUR proteins.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

SOX9 is dispensable for the initiation of epigenetic remodeling and the activation of marker genes at the onset of chondrogenesis.

SOX9 controls cell lineage fate and differentiation in major biological processes. It is known as a potent transcriptional activator of differentiation-specific genes, but its earliest targets and its contribution to priming chromatin for gene activation remain unknown. Here, we address this knowledge gap using chondrogenesis as a model system. By profiling the whole transcriptome and the whole epigenome of wild-type and Sox9-deficient mouse embryo limb buds, we uncover multiple structural and regulatory genes, including Fam101a, Myh14, Sema3c and Sema3d, as specific markers of precartilaginous condensation, and we provide evidence of their direct transactivation by SOX9. Intriguingly, we find that SOX9 helps remove epigenetic signatures of transcriptional repression and establish active-promoter and active-enhancer marks at precartilage- and cartilage-specific loci, but is not absolutely required to initiate these changes and activate transcription. Altogether, these findings widen our current knowledge of SOX9 targets in early chondrogenesis and call for new studies to identify the pioneer and transactivating factors that act upstream of or along with SOX9 to prompt chromatin remodeling and specific gene activation at the onset of chondrogenesis and other processes.

Loss of myosin VI expression affects acrosome/acroplaxome complex morphology during mouse spermiogenesis†.

During spermiogenesis in mammals, actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical, and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures.

The role of MAP-kinase p38 in the m. soleus slow myosin mRNA transcription regulation during short-term functional unloading.

The unloading of postural muscles leads to the changes in myosins heavy chains isoforms (MyHCs) mRNAs transcription pattern, that cause severe alterations of muscle functioning. Several transcription factors such as NFATc1 and TEAD1 upregulate slow MyHC mRNA transcription, and p38 MAP kinase can phosphorylate NFAT and TEAD1, causing their inactivation. However, the role p38 MAP kinase plays in MyHCs mRNAs transcription regulation in postural soleus muscle during unloading remains unclear. We aimed to investigate whether pharmacological inhibition of p38 MAPK during rat soleus unloading would prevent the unloading-induced slow-type MyHC mRNA transcription decrease by affecting calcineurin/NFATc1 or TEAD1 signaling. Male Wistar rats were randomly assigned to three groups: cage control (C), 3-day hindlimb suspended group (3HS) and 3-day hindlimb suspended group with the daily oral supplementation of 10 mg/kg p38 MAPK inhibitor VX-745 (3HS + VX-745). 3 days of hindlimb suspension caused the significant decreases of slow MyHC and slow-tonic myh7b mRNAs transcription as well as the decrease of NFATc1-dependent MCIP1.4 mRNA transcription in rat soleus muscles compared to the cage control. P38 MAP-kinase inhibition during hindlimb suspension completely prevented slow MyHC mRNA content decrease and partially prevented slow-tonic myh7b and MCIP1.4 mRNAs transcription decreases compared to the 3HS group. We also observed NFATc1 and TEAD1 myonuclear contents increases in the 3HS + VX-745 group compared to both 3HS and C groups (p < 0.05). Therefore, we found that p38 inhibition counteracts the unloading-induced slow MyHC mRNA transcription downregulation and leads to the activation of calcineurin/NFAT signaling cascade in unloaded rat soleus muscles.

Promoters to Study Vascular Smooth Muscle.

Smooth muscle cells (SMCs) are a critical component of blood vessel walls that provide structural support, regulate vascular tone, and allow for vascular remodeling. These cells also exhibit a remarkable plasticity that contributes to vascular growth and repair but also to cardiovascular pathologies, including atherosclerosis, intimal hyperplasia and restenosis, aneurysm, and transplant vasculopathy. Mouse models have been an important tool for the study of SMC functions. The development of smooth muscle-expressing Cre-driver lines has allowed for exciting discoveries, including recent advances revealing the diversity of phenotypes derived from mature SMC transdifferentiation in vivo using inducible CreER T2 lines. We review SMC-targeting Cre lines driven by the Myh11, Tagln, and Acta2 promoters, including important technical considerations associated with these models. Limitations that can complicate study of the vasculature include expression in visceral SMCs leading to confounding phenotypes, and expression in multiple nonsmooth muscle cell types, such as Acta2-Cre expression in myofibroblasts. Notably, the frequently employed Tagln/ SM22α- Cre driver expresses in the embryonic heart but can also confer expression in nonmuscular cells including perivascular adipocytes and their precursors, myeloid cells, and platelets, with important implications for interpretation of cardiovascular phenotypes. With new Cre-driver lines under development and the increasing use of fate mapping methods, we are entering an exciting new era in SMC research.

Myosin Heavy Chain Composition, Creatine Analogues, and the Relationship of Muscle Creatine Content and Fast-Twitch Proportion to Wilks Coefficient in Powerlifters.

Machek, SB, Hwang, PS, Cardaci, TD, Wilburn, DT, Bagley, JR, Blake, DT, Galpin, AJ, and Willoughby, DS. Myosin heavy chain composition, creatine analogues, and the relationship of muscle creatine content and fast-twitch proportion to Wilks coefficient in powerlifters. J Strength Cond Res 34(11): 3022-3030, 2020-Little data exist on powerlifting-specific skeletal muscle adaptations, and none elucidate sex differences in powerlifters. Powerlifters tend to display higher fast-twitch fiber content and phosphagen system dependence. Nevertheless, it is unknown whether fast-twitch fiber or muscle creatine content are predictive of competitive powerlifting performance (via Wilks coefficient). Twelve actively competing powerlifters (PL; n = 6M/6F; age = 21.3 ± 1.0; 3.0 ± 1.8 year competing; 7.3 ± 6.6 meets attended) and 10 sedentary controls (CON; n = 5M/5F; age = 19.4 ± 2.0 year) underwent vastus lateralis muscle biopsies and venipuncture to compare the myosin heavy chain (MHC) fiber type and creatine analogue profiles between groups of both sexes, and determine whether MHC IIa and muscle total creatine (MTC) composition predict powerlifting performance. Samples were analyzed for specific MHC isoform (I, IIa, and IIx) content via mixed homogenate SDS-PAGE, and creatine analogues (MTC, muscle creatine transporter [SLC6A8], serum total creatine [STC], and serum creatinine [CRT]). Furthermore, MHC IIa and MTC content were compared with Wilks coefficient using Pearson correlation coefficients. Male PL MHC content was 50 ± 6% I, 45 ± 6% IIa, and 5 ± 11% IIx, versus 46 ± 6% I, 53 ± 6 IIa, and 0% IIx in female PL. Conversely, male CON MHC content was 33 ± 5% I, 38 ± 7% IIa, and 30 ± 8% IIx, vs. 35 ± 9% I, 44 ± 8% IIa, and 21 ± 17% IIx in female CON. Muscle total creatine, SLC6A8, STC, and CRT did not significantly differ between groups nor sexes. Finally, neither MHC IIa content (r = -0.288; p = 0.364) nor MTC (r = 0.488; p = 0.108) significantly predicted Wilks coefficient, suggesting these characteristics alone do not determine powerlifting skill variation.

Classifying AML patients with inv(16) into high-risk and low-risk relapsed patients based on peritransplantation minimal residual disease determined by CBFß/MYH11 gene expression.

Ninety acute myeloid leukemia (AML) patients with inv(16) were monitored CBFß/MYH11 transcript around allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 23 patients received HLA-matched sibling donor transplantation (MSDT) and 67 patients received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were analyzed in this study. Patients were divided into four groups based on CBFß/MYH11 expression prior to transplantation (pre-MRD): with negative (group 1)/positive (group 2) pre-MRD before MSDT; with negative (group 3)/positive (group 4) pre-MRD before haplo-HSCT. The results showed that patients in group 2 had the highest cumulative incidence of relapse (2-year CIR, 40.7%), the lowest leukemia-free survival (2-year LFS, 50.8%), and overall survival (2-year OS, 62.5%). The other three groups of patients had comparable outcomes. The patients were also classified into the other three groups according to CBFß/MYH11 value of + 1 month after transplantation: group 5: pre- and post-transplant MRD were both negative; group 6: the value of post-transplant MRD was lower than 0.2%; group 7: the value of post-transplant MRD was higher than 0.2%. Group 7 had the highest CIR and the lowest LFS. These results indicated that AML patients with inv(16) were able to be separated into high-risk and low-risk relapse groups based on peritransplant MRD determined by RQ-PCR-based CBFß/MYH11. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT.

Time-dependent functional, morphological, and molecular changes in diabetic bladder dysfunction in streptozotocin-induced diabetic mice.

AIM: Diabetic bladder dysfunction (DBD) is one of the most common and bothersome complications of diabetes mellitus (DM). This study aimed to investigate the functional, structural, and molecular changes of the bladder at 0, 3, 6, 9, and 12 weeks after DM induction by streptozotocin (STZ) in male C57BL/6 mice. METHODS: Male C57BL/6J mice were injected with STZ (130 mg/kg). Then, diabetic general characteristics, cystometry test, histomorphometry, and contractile responses to α, ß-methylene ATP, KCl, electrical-field stimulation, carbachol were performed at 0, 3, 6, 9, and 12 weeks after induction. Finally, protein and messenger RNA (mRNA) expressions of myosin Va and SLC17A9 were quantified. RESULTS: DM mice exhibited lower body weight, voiding efficiency and higher water intake, urine production, fasting blood glucose, oral glucose tolerance test, bladder wall thickness, maximum bladder capacity, residual volume, bladder compliance. In particular, nonvoiding contractions has increased more than five times at 6 weeks. And the amplitudes of spontaneous activity, contractile responses to all stimulus was about two times higher at 6 weeks but cut almost in half at 12 weeks. The protein and mRNA expressions of myosin Va and SLC17A9 were about two times higher at 6 weeks, but myosin Va was reverted nearly 40% while SLC17A9 is still higher at 12 weeks. CONCLUSIONS: DBD transitioned from a compensated state to a decompensated state in STZ-induced DM mice at 9 to 12 weeks after DM induction. Our molecular data suggest that the transition may be closely related to the alterations of myosin Va and SLC17A9 expression levels in the bladder with time.

Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.

We aimed to understand the genetic control of cardiac remodeling using an isoproterenol-induced heart failure model in mice, which allowed control of confounding factors in an experimental setting. We characterized the changes in cardiac structure and function in response to chronic isoproterenol infusion using echocardiography in a panel of 104 inbred mouse strains. We showed that cardiac structure and function, whether under normal or stress conditions, has a strong genetic component, with heritability estimates of left ventricular mass between 61% and 81%. Association analyses of cardiac remodeling traits, corrected for population structure, body size and heart rate, revealed 17 genome-wide significant loci, including several loci containing previously implicated genes. Cardiac tissue gene expression profiling, expression quantitative trait loci, expression-phenotype correlation, and coding sequence variation analyses were performed to prioritize candidate genes and to generate hypotheses for downstream mechanistic studies. Using this approach, we have validated a novel gene, Myh14, as a negative regulator of ISO-induced left ventricular mass hypertrophy in an in vivo mouse model and demonstrated the up-regulation of immediate early gene Myc, fetal gene Nppb, and fibrosis gene Lgals3 in ISO-treated Myh14 deficient hearts compared to controls.

Divergent effects of cold water immersion versus active recovery on skeletal muscle fiber type and angiogenesis in young men.

Resistance training (RT) increases muscle fiber size and induces angiogenesis to maintain capillary density. Cold water immersion (CWI), a common postexercise recovery modality, may improve acute recovery, but it attenuates muscle hypertrophy compared with active recovery (ACT). It is unknown if CWI following RT alters muscle fiber type expression or angiogenesis. Twenty-one men strength trained for 12 wk, with either 10 min of CWI ( n = 11) or ACT ( n = 10) performed following each session. Vastus lateralis biopsies were collected at rest before and after training. Type IIx myofiber percent decreased ( P = 0.013) and type IIa myofiber percent increased with training ( P = 0.012), with no difference between groups. The number of capillaries per fiber increased from pretraining in the CWI group ( P = 0.004) but not the ACT group ( P = 0.955). Expression of myosin heavy chain genes ( MYH1 and MYH2), encoding type IIx and IIa fibers, respectively, decreased in the ACT group, whereas MYH7 (encoding type I fibers) increased in the ACT group versus CWI ( P = 0.004). Myosin heavy chain IIa protein increased with training ( P = 0.012) with no difference between groups. The proangiogenic vascular endothelial growth factor protein decreased posttraining in the ACT group versus CWI ( P < 0.001), whereas antiangiogenic Sprouty-related, EVH1 domain-containing protein 1 protein increased with training in both groups ( P = 0.015). Expression of microRNAs that regulate muscle fiber type (miR-208b and -499a) and angiogenesis (miR-15a, -16, and -126) increased only in the ACT group ( P < 0.05). CWI recovery after each training session altered the angiogenic and fiber type-specific response to RT through regulation at the levels of microRNA, gene, and protein expression.

BGP-15 improves contractile function of regenerating soleus muscle.

This study investigated the effect of the heat shock protein inducer O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic amidoxime (BGP-15) on the morphology and contractile function of regenerating soleus muscles from mice. Cryolesioned soleus muscles from young mice treated daily with BGP-15 (15 mg/Kg) were evaluated on post-cryolesion day 10. At this time point, there was a significant decrease in the cross-sectional area of regenerating myofibers, maximal force, specific tetanic force, and fatigue resistance of regenerating soleus muscles. BGP-15 did not reverse the decrease in myofiber cross-sectional area but effectively prevented the reduction in tetanic force and fatigue resistance of regenerating muscles. In addition, BGP-15 treatment increased the expression of embryonic myosin heavy chain (e-MyHC), MyHC-II and MyHC-I in regenerating muscles. Although BGP-15 did not alter voltage dependent anion-selective channel 2 (VDAC2) expression in cryolesioned muscles, it was able to increase inducible 70-kDa heat shock protein (HSP70) expression. Our results suggest that BGP-15 improves strength recovery in regenerating soleus muscles by accelerating the re-expression of adult MyHC-II and MyHC-I isoforms and HSP70 induction. The beneficial effects of BGP-15 on the contractile function of regenerating muscles reinforce the potential of this molecule to be used as a therapeutic agent.

Apigenin inhibits sciatic nerve denervation-induced muscle atrophy.

INTRODUCTION: Apigenin (AP) has been reported to elicit anti-inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation-induced muscle atrophy. METHODS: Sciatic nerve-denervated mice were fed a 0.1% AP-containing diet for 2 weeks. Muscle weight and cross-sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. RESULTS: Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis-alpha in the gastrocnemius and soleus muscles, and interleukin-6 expression in the soleus muscle. DISCUSSION: AP appears to inhibit denervation-induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58: 314-318, 2018.

Cytosolic proteome profiling of monocytes for male osteoporosis.

In male Caucasians with discordant hip bone mineral density (BMD), we applied the subcellular separation and proteome profiling to investigate the monocytic cytosol. Three BMD-associated proteins (ALDOA, MYH14, and Rap1B) were identified based on multiple omics evidence, and they may influence the pathogenic mechanisms of osteoporosis by regulating the activities of monocytes. INTRODUCTION: Osteoporosis is a serious public health problem, leading to significant mortality not only in aging females but also in males. Peripheral blood monocytes (PBMs) play important roles in bone metabolism by acting as precursors of osteoclasts and producing cytokines important for osteoclast development. The first cytosolic sub-proteome profiling analysis was performed in male PBMs to identify differentially expressed proteins (DEPs) that are associated with BMDs and risk of osteoporosis. METHODS: Here, we conducted a comparative proteomics analysis in PBMs from Caucasian male subjects with discordant hip BMD (29 low BMD vs. 30 high BMD). To decrease the proteome complexity and expand the coverage range of the cellular proteome, we separated the PBM proteome into several subcellular compartments and focused on the cytosolic fractions, which are involved in a wide range of fundamental biochemical processes. RESULTS: Of the total of 3796 detected cytosolic proteins, we identified 16 significant (P < 0.05) and an additional 22 suggestive (P < 0.1) DEPs between samples with low vs. high hip BMDs. Some of the genes for DEPs, including ALDOA, MYH14, and Rap1B, showed an association with BMD in multiple omics studies (proteomic, transcriptomic, and genomic). Further bioinformatics analysis revealed the enrichment of DEPs in functional terms for monocyte proliferation, differentiation, and migration. CONCLUSIONS: The combination strategy of subcellular separation and proteome profiling allows an in-depth and refined investigation into the composition and functions of cytosolic proteome, which may shed light on the monocyte-mediated pathogenic mechanisms of osteoporosis.

Intrinsic MYH7 expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy.

HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the ß-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.

Noninvasive Assessment of Skeletal Muscle Myosin Heavy Chain Expression in Trained and Untrained Men.

Fry, AC, Housh, TJ, Cramer, JB, Weir, JP, Beck, TW, Schilling, BK, Miller, JD, and Nicoll, JX. Noninvasive assessment of skeletal muscle myosin heavy chain expression in trained and untrained men. J Strength Cond Res 31(9): 2355-2362, 2017-Numerous conditions and types of physical activity (e.g., exercise, aging, and muscle-related diseases) can influence muscle fiber types and the proteins expressed. To date, muscle fibers can only be characterized by actually obtaining a tissue sample using the invasive muscle biopsy procedure. Mechanomyography (MMG) is the assessment of the vibration properties of contracting skeletal muscle and has been proposed as a possible noninvasive method for muscle fiber analysis. Therefore, the purpose of this project was to examine the feasibility of using MMG and muscle performance measures to noninvasively assess muscle fiber characteristics. Fifteen men (5 endurance-trained, 5 weight-trained, and 5 sedentary) provided muscle samples from their vastus lateralis muscle. These samples were analyzed for relative myosin heavy chain (MHC) protein expression, which is highly correlated with % muscle fiber type areas. Additionally, each subject performed several muscle performance tests, and MMG of the quadriceps was assessed during a knee extension exercise. Multiple regression was used to develop prediction equations for determining relative muscle content of MHC types I, IIa, and IIx. A combination of MMG and knee extension performance variables estimated types I, IIa, and IIx MHCs with approximately 80% accuracy. Although preliminary, these data suggest that muscle performance tests in addition to MMG assessments during a simple muscle performance task (knee extension) can be used to estimate muscle fiber type composition in a healthy male population. Such methods could ultimately be used to noninvasively monitor muscle health and fitness.

A new role for the calcineurin/NFAT pathway in neonatal myosin heavy chain expression via the NFATc2/MyoD complex during mouse myogenesis.

The calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. Here, we show that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. We found this cooperation to be crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, we identified NFATc2 bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. We provide evidence that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, our findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis.

Stiff matrix induces switch to pure ß-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Bovine (Bos taurus) Bone Marrow Mesenchymal Cell Differentiation to Adipogenic and Myogenic Lineages.

PURPOSE: We evaluated the effect of peroxisome proliferator-activated receptor (PPAR) agonists on the differentiation and metabolic features of bovine bone marrow-derived mesenchymal cells induced to adipogenic or myogenic lineages. METHODS: Cells isolated from 7-day-old calves were cultured in basal medium (BM). For adipogenic differentiation, cells were cultured for one passage in BM and then transferred to a medium supplemented with either rosiglitazone, telmisartan, sirtinol or conjugated c-9, t-11 linoleic acid; for myogenic differentiation, third-passage cells were added with either bezafibrate, telmisartan or sirtinol. The expression of PPARx03B3; (an adipogenic differentiation marker), myosin heavy chain (MyHC; a myogenic differentiation marker) and genes related to energy metabolism were measured by quantitative real-time PCR in a completely randomized design. RESULTS: For adipogenic differentiation, 20 µM telmisartan showed the highest PPARx03B3; expression (15.58 ± 0.62-fold, p < 0.0001), and differences in the expression of energy metabolism-related genes were found for hexokinase II, phosphofructokinase, adipose triglyceride lipase, acetyl-CoA carboxylase α(ACACα) and fatty acid synthase (p < 0.001), but not for ACACß (p = 0.4275). For myogenic differentiation, 200 µM bezafibrate showed the highest MyHC expression (73.98 ± 11.79-fold), and differences in the expression of all energy metabolism-related genes were found (p < 0.05). CONCLUSIONS: Adipocyte and myocyte differentiation are enhanced with telmisartan and bezafibrate, respectively, and energy uptake, storage and mobilization are improved with both.

Knockdown of Myosin 6 inhibits proliferation of oral squamous cell carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) accounts for 95% of all oral cancer with higher mortality and morbidity rates worldwide. However, the potential molecular mechanism of OSCC remains largely unclear. Myosin VI (MYO6) is a unique actin motor and reported to be overexpressed in several cancers. This study aims to examine the functional relationship between OSCC and MYO6. METHODS: The mRNA expression of MYO6 was firstly investigated by analyzing data derived from Oncomine database. On the basis of the results, the expression of MYO6 was knocked down using lentivirus-delivered RNA interference in human OSCC cell line CAL27, as confirmed by qPCR and Western blot analysis. Stable MYO6 knockdown cells were employed to determine the effects of MYO6-silencing on cell growth by MTT, colony formation and cell cycle distribution and apoptosis by flow cytometry assay. Moreover, the expressions of cell apoptotic proteins were examined by Western blot analysis. RESULTS: We first observed MYO6 was overexpressed in tongue squamous cell carcinoma TSCC belongs to OSCC, compared with normal tissues. For cellular analysis, shRNA sequences against MYO6 could efficiently reduce its expression in CAL27 cells. Knockdown of MYO6 significantly decreased cell proliferation, caused cell cycle arrest at G2/M phase, and promoted cell apoptosis. Moreover, cell apoptosis-associated proteins, caspase-3 and PARP, were obviously upregulated in CAL27 after MYO6-silencing. CONCLUSION: MYO6 could play an essential role in the growth of OSCC cells via regulation of cell cycle progression and apoptosis.

Comparisons of different myosin heavy chain types, AMPK, and PGC-1α gene expression in the longissimus dorsi muscles in Bama Xiang and Landrace pigs.

Bama Xiang and Landrace pigs are the local fatty and lean breeds, respectively, in China. We compared differences in carcass traits, meat quality traits, and myosin heavy chain (MyHC) types in the longissimus dorsi muscles between Bama Xiang and Landrace pigs. This was done in pigs of the same age, using real-time PCR, to investigate the relationship between MyHC fiber types and carcass characteristics, meat quality traits, and the key factors regulating muscle fiber type. Bama Xiang pigs exhibited smaller size and slower growth than Landrace pigs (P < 0.01). We found that the superior meat quality, especially the high intramuscular fat (IMF) content in Bama Xiang pig, was related to elevated type I oxidative muscle fiber content (P < 0.01). In contrast, Landrace pig muscle had a higher glycolytic type IIb muscle fiber content (P < 0.01). MyHC I gene expression was significantly positively correlated with backfat thickness and IMF content (P < 0.01). MyHC IIb was significantly negatively correlated with IMF content (P < 0.05), and positively correlated with carcass yield (P < 0.05). AMP-activated protein kinase and peroxisome proliferator-activated receptor-g coactivator-1a are suggested to be the two key factors regulating muscle fiber type in pigs. Our results indicate that muscle fiber composition is one of the key differences leading to the differences of meat quality between Bama Xiang and Landrace pigs. These results may provide a theoretical basis for further studies of the molecular mechanism underlying the excellent meat quality of the Bama Xiang pig.