Integrity Of Myofibrils Research Articles

Physiological, biochemical, and histological changes in trout muscles as a result of white lupine inclusion in the diet structure

The work evaluated the effect of white lupin on the physiological, biochemical, and histological parameters of rainbow trout. The data of water chemical analysis, biochemical and histological analysis of rainbow trout muscles are presented. The results of the experiment indicate more pronounced positive results in the experimental groups, the average live weight of trout at the end of the studies in the 2nd experimental group was 3.7% higher relative to the control group. The average live weight of the 3rd group was 18.5% higher and that of the 4th group was 7.4%. At the end of the studies, an increase in muscle mass was observed in group 2 by 1.5% more than in the control group, group 3 – 18.8% and in the 4 experimental group – 12.8%, respectively. According to the results of biochemical analysis of muscle tissue, an increase in protein content in the experimental groups was established, in 2 experimental group by 0.2%, in 3 experimental group – 3.59%, and in 4 experimental group – 3.11%. The fat content in the muscle tissue of rainbow trout of the experimental groups decreased by 0.46% in 2 experimental group, 1.0% in 3 experimental group, and 0.96% in 4 experimental group. Therefore, the use of white lupin in the diet as a high-protein component affects the reduction of body fat in rainbow trout and contributes to an increase in protein content. Histological analysis of the white muscles of the control and experimental groups of trout showed moderate separation of muscle fibers and integrity of myofibrils. The striation of the transverse muscle was well pronounced. The experiment established a positive effect on its productivity, the biochemical composition of muscle tissue, and the quality of the products obtained.

High-Intensity Ultrasonication as an Innovative Approach for the Softening of Wooden Breast Meat in Broilers

Considering the remarkable implications exerted by the occurrence of wooden breast (WB) abnormality on meat tenderness and marketability, the poultry processing industry demands the implementation of postmortem procedures that can improve the textural traits of chicken breasts affected by this defect. Within this scenario, this study aimed at exploring the effectiveness of high-intensity ultrasonication (HIU) in attenuating the toughness of WB fillets and evaluating its effects on the main technological properties and quality characteristics of chicken breast meat. Overall outcomes showed that HIU significantly reduced (P<0.01) the compression forces of both unaffected and WB raw meat without negatively affecting the main meat quality traits and technological properties, such as color and water holding capacity. The significant (P<0.05) increase in myofibrillar fragmentation index (MFI) observed in unaffected fillets might hint at an alteration of myofibril integrity following the mechanical action of ultrasonic waves. However, despite the effectiveness of HIUin improving the textural traits of raw WB meat, overall outcomes obtained through western blot and MFI analyses suggested that HIU did not remarkably alter the microstructure of myopathic muscles. The myodegenerative lesions typically occurring in WB muscles may have partially disguised the mechanical effects of ultrasonic waves on muscle cells’ structures, making the elucidation of the mechanisms that lead to the softening of WB meat particularly complex.

Age-related changes of myofibrils in white laboratory rats of different ages

The study of isolated preparations of experimental white rats of 14 months and older reveals that the longitudinal orientation of cardiomyocytes is replaced by an angular orientation in older mice. The number and structural integrity of myofibrils occupy one of the leading places in the regulation of the contraction-relaxation cycle and the implementation of the adaptive capabilities of the myocardium in the normal functioning of cardiomyocytes. Our experimental studies aim at describing the quantitative and morphological characteristics of myofibrils in rats of two age groups and analyzing the morphological and functional results. Isolated heart preparations were examined in two age groups of white rats (middle - 8-14 years; elderly - 14 months and more. The number of rats in each group amounted to six) using different histological staining methods. The results obtained show that, compared with middle-aged rats, the development of degenerative changes in myofibrils is observed in old rats, which is manifested in structural inferiority and quantitative reduction. Degenerative changes revealed with age at the intracellular level can be considered an important morphological basis for the formation of functional heart failure and the depletion of adaptive capabilities.

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure.

Simple SummaryMyosin is a molecular motor protein that is critical for using stored energy to yield contraction in both skeletal muscle and in the heart. Mutations in myosin can result in inherited human hypertrophic cardiomyopathy, wherein the heart walls become thickened. This results in inability to adequately pump enough oxygenated blood to meet bodily demands, and can lead to abnormal heart beating patterns and the possibility of sudden death. Typically, over-active myosin can be causative for this disease. Here, we used the common fruit fly to study one of these cardiomyopathy-causing mutations by expressing the mutant protein in the indirect flight muscle of the fly. Surprisingly, we found that the mutation and associated mutations decrease myosin and muscle function instead of enhancing it. We show that at least some of these defects result from improper interactions within the myosin protein. This suggests that appropriate communications within myosin are critical for its function and further indicates that certain aspects of indirect flight muscle myosin are functionally distinct from those of myosin in the human heart. By understanding the intricate interactions within the myosin motor protein that are important for its normal role, therapeutics for improving mutant myosin function can be developed.The R249Q mutation in human β-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a β-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent β-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the β-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

Evaluation of ultrasound-assisted L-histidine marination on beef M. semitendinosus: Insight into meat quality and actomyosin properties

This paper aimed to evaluate the effects of ultrasound-assisted L-histidine marination (UMH) on meat quality and actomyosin properties of beef M. semitendinosus. Our results found that UMH treatment effectively avoided excessive liquid withdrawal, and disrupted myofibril integrity by modifying the water distribution and weakening connection of actin-myosin with increased muscle pH. The ultrasound-treated sample provided more opportunity for the filtration of L-histidine to intervene the isoelectric point and conformation of muscle protein. The activated caspase-3 and changes of ATPase activity in UMH-treated meat accelerated the postmortem ageing, and L-histidine might competitively inhibit the actin-myosin binding by the imidazole group. UMH decreased the surface hydrophobicity by shielding hydrophobic area and unfolding the actomyosin structure. In addition, the increased actomyosin solubility with smaller particle size enhanced the SH content for better cross-linking of myosin tail, and formation of heat-set gelling protein structure. Therefore, UMH treatment manifested the potential to improve beef quality.

Calmodulin interactions within the N2A region of titin

One of the most interesting elastic proteins is the muscle protein titin, which exists in the sarcomere of the muscle and plays a critical role in maintaining myofibril integrity and in passive force. Within titin, there are several types of structural elements that work synergistically to produce the various functions of the protein. Our lab has recently been focused on the N2A region, which is located in the center of the I-band region of titin. Structurally, the N2A region is comprised of four immunoglobulin domains and a unique region that consists of 117 amino acids in both ordered and disordered regions.

Texture and Quality Assessment of Ready-to-eat Farmed Obscure Puffer Fish (Takifugu obscurus) Fillet by Evaluating Bacterial and Myofibrillar Degradation and Biochemical Changes during Refrigerated Storage

ABSTRACT Obscure puffer fish (Takifugu obscurus) flesh, often eaten as raw fish fillet, is susceptible to softening. To investigate the mechanisms for the softening of flesh and changes in firmness, the proliferation of total microbial flora, Enterobacteriaceae, Pseudomonas spp., and lactic acid bacteria (LAB), total volatile basic nitrogen (TVBN), trimethylamine (TMA), and pH were periodically assayed during storage at 0, 4, and 8ºC. Myofibril extraction and separation of puffer fish flesh was carried out and the length of the separated myofibrils was measured periodically using an optical microscope during the entire storage time. Transmission electron microscopy was also used to inspect the changes in myofibrillar characteristics of the fish flesh at the beginning and end of the storage period. The results indicated that lower chilled temperature can contribute to reduced proliferation of microbial flora and less variation in TVBN, TMA, and pH and further maintain the integrity of myofibrils and retain firmness of puffer fish flesh during storage.

Identification of KIAA0196 as a novel susceptibility gene for myofibril structural disorganization in cardiac development

BackgroundCongenital heart disease is one of the most common cardiac malformation-related diseases worldwide. Some causative genes have been identified but can explain only a small proportion of all cases; therefore, the discovery of novel susceptibility genes and/or modifier genes for abnormal cardiac development remains a major challenge. MethodsWe used a single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS) was conducted to screen and quickly identify candidate genes. KIAA0196 knockout zebrafish and mice were generated by CRISPR/Cas9 to detect whether or how KIAA0196 deficiency would influence cardiac development. ResultsHomozygous, but not heterozygous, zebrafish and mice showed early embryonic lethality. At the embryonic stage, microscopic examination and dissection revealed pericardial edema and ventricle enlargement in homozygous zebrafish and obviously delayed cardiac development in heterozygous mice, while echocardiography and tissue staining showed that significantly decreased cardiac function, ventricle enlargement, myofibril loss, and significantly reduced trabecular muscle density were observed in adult heterozygous zebrafish and mice. Most importantly, immunostaining and electron microscopy showed that there was a significant increase in sarcomere structural disorganization and myofibril structural integrity loss in KIAA0196 mutants. Furthermore, substantial downregulation in other sarcomeric genes and proteins was detected and verified in a mouse model via transcriptome and proteomics analyses; these changes especially affected the myosin heavy or light chain (MYH or MYL) family genes. ConclusionWe identified KIAA0196 for the first time as a susceptibility gene for abnormal cardiac development. KIAA0196 deficiency may cause abnormal heart development by influencing the structural integrity of myofibrils.

Restrictive cardiomyopathy: difficulties desminopathy diagnostics

The article provides a brief overview of the problems of diagnostics and etiological verification of restrictive cardiomyopathy (RCMP). Multiple causes lead to the restrictive phenotype of intracardiac hemodynamics and diastolic dysfunction of the heart: infiltrative (amyloidosis, sarcoidosis, elastoma, metabolic syndrome, tumor metastasis to the myocardium); endomyocardial (tropical endomyocardial fibrosis, hypereosinophilic syndrome, carcinoid, radiation damage); accumulation diseases (hemochromatosis, Pompe and Gaucher diseases, Fabry disease). RKMP is also characteristic for desminopathy — so-called desmin storage disease. This is a disease caused by mutations in the desmin gene (DES) that determine the development of cardiomyopathy or myofibrillar myopathy, and in some cases cause a combined phenotype of the disease. Desmin belongs to the group of intermediate filaments that maintain the structural and functional integrity of myofibrils. Mutations in DES lead to disruption of the assembly of filaments and a change in the myofibrillar cell lattice, which contributes to an increased vulnerability of myocytes to mechanical stress. The article presents a clinical case of familial desminopathy associated with a new missense R118P mutation of DES gene, characterized by dominant cardiac RCMP phenotype and subclinical manifestations of myofibrillar myopathy.

Characterization of Breast Meat Collected from Spent Lohmann Brown Layers in Comparison to Commercial Ross Broilers

ABSTRACT The objective of this study was to compare characteristics and properties of breast meat from spent Lohmann Brown layers (SP, 90 weeks old, n = 24) and those of commercial broilers (BR, 6 weeks old, n = 24). The breasts of both SP and BR were collected from a local processing plant, vacuum-packed in a plastic bag, and stored at -18oC until further analyses. The SP showed a greater water-holding capacity, tougher and chewier texture (p<0.05). Both raw and cooked SP breasts comprised the greater total collagen but lower soluble collagen than those of BR (p<0.05). Muscle fibers of the SP, observed under scanning electron microscope, were small, with average fiber diameter of 37.85±1.40 μm, densely packed and surrounded by complex networks of connective tissue. The lower myofibril fragmentation index and alkaline-soluble protein fractions of the SP (p<0.05) suggested lesser degree of postmortem fragmentation and protein denaturation in SP compared to BR. Based on differential scanning calorimetry, multiple endothermic transitions were observed in both raw BR and SP breast samples. While BR thermograms comprise five transitions at 57.7oC, 64.2oC, 67.6oC, 72.3oC and 77.6oC with total ΔH of 15.31 J/g dry meat, the SP samples exhibited four transitions at 55.1oC, 62.1oC, 70.5oC and 77.7oC and total ΔH of 17.62 J/g dry meat. Overall, the findings indicated that cooked meat toughness of SP was attributed by the high total and heat-stable collagen content, densely packing of small muscle fibers, and the superior myofibril integrity.

SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

Reactive oxygen species (ROS) contribute to the etiology of multiple muscle-related diseases. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is incompletely understood how cellular stress induces structural destabilization of sarcomeres. Here we report that glutathionylation of SMYD2 contributes to a loss of myofibril integrity and degradation of sarcomeric proteins mediated by MMP-2 and calpain 1. We used a clickable glutathione approach in a cardiomyocyte cell line and found selective glutathionylation of SMYD2 at Cys13. Biochemical analysis demonstrated that SMYD2 upon oxidation or glutathionylation at Cys13 loses its interaction with Hsp90 and N2A, a domain of titin. Upon dissociation from SMYD2, N2A or titin is degraded by activated MMP-2, suggesting a protective role of SMYD2 in sarcomere stability. Taken together, our results support that SMYD2 glutathionylation is a novel molecular mechanism by which ROS contribute to sarcomere destabilization.

Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis.

Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-β1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-β1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-β1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFβ-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFβ/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signalling leads to chronic fibrosis.

Myofibrilar functional dysregulation in fish: A new biomarker of damage to pesticides

Endosulfan (ES) modifies the ultrastructure of skeletal muscle fibers and causes changes to the swimming behavior of fish. The objectives of the present work were to evaluate, in fishes of Australoheros facetus, 1) the integrity of myofibrils (Mf) by the analysis of SDS-PAGE profiles, and 2) the functionality of Mf through the microscopically monitoring of the contraction and changes in Mg2+ Ca2+- ATPase and Mg2+(EGTA) -ATPase activities. As expected, after the addition of the contraction buffer, control fish Mf contracted. On the contrary, Mf from fish exposed at 0.5 μg/L ES showed a partial contraction and none of the fish exposed at 10 μg/L ES contracted. As judged by its high Mg2+ Ca2+ ATPase activity and low Mg2+ (EGTA) ATPase activity, control Mf showed good functionality. In Mf from fish exposed to 0.5 and 10 μg/L ES the activities of these enzymes were similar, suggesting denaturation or degradation of some component of tropomyosin–troponin complex. SDS-PAGE patterns of Mf from fish exposed to ES showed degradation of the myosin heavy chain and of tropomyosin. Similar values of lipid peroxidation (TBARS) were found in both control and exposed Mf, suggesting that lipid oxidation was not be related to the above-mentioned changes. The observed effects expand the knowledge of ES action in muscles and could be used as biomarkers of damage in fishes exposed to organochlorine compounds like the insecticide endosulfan.

Structure of northern snakehead (Channa argus) meat: Effects of freezing method and frozen storage

ABSTRACTThis study was conducted to investigate the potential of cryogenic freezing with liquid nitrogen in the shelf-life extension of northern snakehead (Channa argus) and clarify the effects of temperature fluctuations after freezing on the quality attributes and tissue microstructure during frozen storage. The fish fillets were frozen by three methods including freezing using an ultra-low-temperature freezer (−80°C) to the core temperature of −60°C (T1) or −18°C (T2), or liquid nitrogen (T3) followed by storage at −20°C for five months. Cryogenic freezing with liquid nitrogen postponed the decrease in pH and protein extractability. Temperature fluctuations after freezing might promote the accretion of ice crystals and resulted in the loss of tissue integrity and disorganization of myofibrils. The microstructural changes contributed greatly to the increased thawing loss and decreased resilience, as indicated by the enlarged extracellular spacing and the flakiness of myofibrils. Cryogenic freezing with liquid nitrogen showed no superiority in maintaining the microstructure of northern snakehead fillets, which was supposedly attributed to the cracking in tissue during freezing and the accretion of ice crystals during frozen storage.

The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes.

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.

Abstract 271: Deletion of Delta-like 1 Homolog Accelerates Fibroblast-myofibroblast Differentiation and Induces Myocardial Fibrosis

Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. Here we uncover that Delta-like homologue 1 (Dlk1), a paternally imprinted gene encoding a transmembrane protein belonging to the Epidermal Growth Factor (EGF)-like family, orchestrates the process of cardiac fibroblast to myofibroblast differentiation and controls myocardial fibrosis. We first show that cardiomyocytes and cardiac fibroblasts express different Dlk1 mRNA spliced variants and its absence accelerates fibroblast differentiation into myofibroblasts in vitro. Overexpression of Dlk1 in cardiac fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process appears to be regulated by TGFβ-1 signaling, since fibroblasts lacking Dlk1 exhibited a higher activation of the TGFβ-1/Smad-3 pathway at baseline, leading to an earlier acquisition of the myofibroblast phenotype. Dlk1-null mice myocardium displayed increased TGFβ-1/Smad3 profibrotic activity, resulting in infiltration/accumulation of myofibroblasts, and induction and deposition of the extracellular matrix fibronectin extra domain A isoform and collagen, supporting a role for Dlk1 in cardiac fibrosis. Furthermore, these profibrotic events were associated with reduced myofibril integrity, myocyte hypertrophy and cardiac dysfunction. Interestingly, Dlk1 expression was downregulated in ischemic heart tissue from human patients and in the border and scar-zones of infarcted pigs’ hearts. This phenotype was paralleled by increased expression of the profibrotic markers, collagen I, lysyl oxidase and α-smooth muscle actin. Mechanistically, the inhibitory action of Dlk1 on cardiac fibroblast-myofibroblast differentiation is mediated by miR-370 direct targeting of TGFβ-R2/Smad-3 signaling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signaling leads to chronic fibrosis.

Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.

Decreased Desmin expression in the developing diaphragm of the nitrofen-induced congenital diaphragmatic hernia rat model.

Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle. DES -/- knockout mice exhibit significant reductions in stiffness and elasticity of the developing diaphragmatic muscle tissue. Furthermore, sequence changes in the DES gene have recently been identified in human cases of CDH, suggesting that alterations in DES expression may lead to diaphragmatic defects. This study was designed to investigate the hypothesis that diaphragmatic DES expression is decreased in fetal rats with nitrofen-induced CDH. Time-mated Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18, and dissected diaphragms (n=72) were divided into control and nitrofen-exposed specimens (n=12 per time-point and experimental group, respectively). Laser-capture microdissection was used to obtain diaphragmatic tissue elements. Diaphragmatic gene expression of DES was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for DES was combined with the mesenchymal marker GATA4 to evaluate protein expression and localization in developing fetal diaphragms. Relative mRNA expression levels of DES were significantly decreased in pleuroperitoneal folds on D13 (1.49±1.79 vs. 3.47±2.32; p<0.05), developing diaphragms on D15 (1.49±1.41 vs. 3.94±3.06; p<0.05) and fully muscularized diaphragms on D18 (2.45±1.47 vs. 5.12±3.37; p<0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of DES mainly in diaphragmatic MCT, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls. Decreased expression of DES in the fetal diaphragm may disturb the basic integrity of myofibrils and the cytoskeletal network during myogenesis, causing malformed MCT and leading to diaphragmatic defects in the nitrofen-induced CDH model.

Abstract 247: Rtf1 Controls Sarcomere Integrity and Cardiac Function

Sarcomere integrity must be continuously maintained in cardiomyocytes to ensure contractile function; however, the genetic programs controlling this process are not completely understood. We have previously shown that the RNA polymerase II associated protein Rtf1 directs cardiac progenitor cell formation during early cardiogenesis. Here we report that in mature cardiomyocytes, Rtf1 controls gene programs essential for the maintenance of sarcomere structure and cardiac function. Cardiomyocyte-specific ablation of Rtf1 in mice results in dilated cardiomyopathy characterized by thinning of the myocardial walls, dilation of the ventricular cavity and impaired left ventricle contractile function. Rtf1 knockdown in neonatal rat ventricular myocytes model results in the breakdown of sarcomeres and downregulation of myofibrillar gene programs accompanied by reduced abundance of monoubiquitinated H2BK120 and epigenetic marks associated with actively transcribed genes. In zebrafish embryos, mutation in Rtf1’s histone modification domain arrests cardiomyocyte differentiation and heart tube formation. Together these findings demonstrate that Rtf1 controls cardiomyocyte maturation, myofibril integrity and cardiac function by modulating cardiac epigenetic landscapes.

Mouse myofibers lacking the SMYD1 methyltransferase are susceptible to atrophy, internalization of nuclei and myofibrillar disarray.

ABSTRACTThe Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6cre produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity.