Project description:In ischemic cardiomyopathy (ICM), left ventricular systolic dysfunction leads to reduced blood flow and oxygen supply to the heart. Alterations in sarcomeric protein function and expression play prominent roles in the onset and progression of cardiomyopathies; however, the molecular mechanisms underlying ICM remain poorly defined. Herein, we have implemented a top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics method for the simultaneous quantification of sarcomeric protein expression and modifications in non-failing donor (n = 16) compared to end-stage failing ICM (n = 16) human cardiac tissues. Our top-down proteomics platform provided a “bird’s eye view” of proteoform families with high mass accuracy and reproducibility. In addition, quantification of post-translational modifications (PTMs) and expression reveal significant changes in various sarcomeric proteins extracted from ICM tissues. Changes include altered phosphorylation and expression of cardiac troponin I (cTnI) and enigma homolog 2 (ENH2) as well as a marked increase in muscle LIM protein (MLP) and calsarcin-1 phosphorylation in ICM hearts. Our results imply that the contractile apparatus of the sarcomere is severely dysregulated during ICM. Thus, this study is the first to uncover significant molecular changes to multiple sarcomeric proteins in end-stage ischemic heart failure patients using LC-MS-based top-down proteomics.
Project description:Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins--the proteins that constitute the contractile apparatuses.
Project description:Oxidative posttranslational modifications (Ox-PTMs) regulate cellular homeostasis in several tissues, including skeletal and cardiac muscles. The putative relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism has not been previously described. We determined the metabolic phenotype and the Ox-PTM profile in the skeletal and cardiac muscles of rats selected for low (LCR) or high (HCR) intrinsic aerobic capacity. The HCR rats have a pronounced increase in mitochondrial content and antioxidant capacity when compared to LCR rats in the skeletal muscle, but only modest changes in the cardiac muscle. Redox proteomics analysis reveals that HCR and LCR rats have different Ox-PTM of cysteine (Cys) residue profile in the skeletal and cardiac muscles. HCR rats have higher number of oxidized Cys residues in the skeletal muscle and conversely display higher number of reduced Cys residues in the cardiac muscle than LCR rats. Most of the proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of the oxidative metabolism. The most significantly oxidized protein in the skeletal muscle of HCR rats is malate dehydrogenase (MDH1). Interestingly, HCR rats show higher MDH1 activity in the skeletal muscle, but not in the cardiac muscle. Thus, this study uncovers an association between Ox-PTMs and intrinsic aerobic capacity, providing new insights into the role of Ox-PTMs as essential signaling to maintain metabolic homeostasis in different muscle types.
Project description:Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca2+-sensitivity and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model. Here, we quantified the phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome.
Project description:Texel and Ujumqin sheep show obvious differences in muscle and fat growth, so they are ideal models not only to understand the molecular mechanism in prenatal skeletal muscle development, but to identify the potential target genes of myostatin. To elucidate the phenotypic variation between the two sheep breeds and the dynamic characteristics of gene expression in skeletal muscle during the development, we examined the development of skeletal muscle in transcriptome-wide level at 70, 85,100,120 , 135 days post coitus (dpc),birth, 1 month and 2 month. Using the specialized and standardized sheep transcriptome-wide oligo DNA microarray (Agilent), we analyzed the transcriptomic profiles of longissmuss dorsi muscle from fetuses of Texel and Ujumqin sheep. We characterized dynamic transcriptome-wide profiles that accompany the prenatal skeletal muscle and fat development in Texel and Ujumqin sheep respectively, and compared the difference in profiles of gene expression between the two sheep breeds at the same developmental stage.Some potential myostatin target genes and other genes controlling the growth of skeletal muscle and adipose were identified for further examinations. Our findings not only contribute to understand the molecular mechanism of prenatal skeletal muscle development in large precocial species, but also provide some clues for human myopathy and obesity at prenatal stages. Moreover, we also can identify putative candidate genes for meat quality traits in farm animals. Longissimus dorsi muscles were sampled from five prenatal development stages (70, 85, 100, 120 and 135 day of gestation) in Texel and eight development stages (at 70, 85, 100, 120, 135 days post coitus (dpc), birth, 1 month and 2 month) in Ujumqin sheep. There were at least three replicates at each development time in each breed. Two gene expression experiments were conducted with a total of 40 hybridizations.
Project description:Skeletal muscles are comprised of gigantic multinucleated cells called muscle fibers, which often span several centimetres in length. Each muscle fiber is densely packed with contractile force-producing myofibrils and ATP-producing mitochondria. During animal development the size of the individual muscle fibers must dramatically increase to match the growth of the animal and to connect growing skeletal elements. How such dramatic tissue growth is coordinated with growth of the contractile apparatus in the muscle fibers is not well understood. Here, we use the large Drosophila flight muscles to mechanistically decipher how muscle fiber growth is controlled during development. We isolated flight muscles from Drosophila melanogaster pupae at 24 h and 32 h APF of the genotypes wt, Dlg5-IR, Slmap-IR, Yorkie-CA, Hippo-IR and isolated total RNA for subsequent BRB-seq sequencing. Our study reveals that regulated activity of core members of the Hippo pathway, Hippo, Warts and Yorkie is required to support post-mitotic flight muscle growth. Interestingly, we identify Dlg5 and Slmap as important members of the STRIPAK phosphatase complex, which negatively regulates Hippo activity and therefore enables post-mitotic muscle growth. Mechanistically, we find that the Hippo pathway controls the timing and the levels of sarcomeric gene expression during muscle development and thus regulates the key components that mediate muscle fiber growth. Since Dlg5, STRIPAK and the Hippo pathway are conserved in mammals a similar mechanism may contribute to skeletal muscle or cardiac growth in humans.
Project description:Microarrays were used for transcription profiling of skeletal muscle samples taken at birth, when the phenotype was not expressed, and 12 weeks of age from Callipyge and wild type sheep. The genes that underlie the expression of the phenotype rather than result from the fibre type change in the affected muscle have been identified. We used microarrays to detail the global programme of gene expression underlying the hypertrophy phenotype and identified distinct classes of regulated genes during this process. A working model that links the muscle hypertrophy phenotype with a core group of transcriptional coregulators is proposed. Experiment Overall Design: Gene expression analyses were performed primarily on longissimus dorsi skeletal muscle (LD) from Wild type (NN) and Callipyge (NCpat) sheep using Bovine Affymetrix GeneChip microarrays. Two developmental time-points were investigated in this study: newborn (within 5 days of birth; T0) and 11-12 weeks post-birth (T12). The muscle hypertrophy phenotype developed over the first 2-3 months and was associated with a significant change in muscle fibre type (Carpenter et al., 1996; Cockett et al., 1994; 1996; Kerth et al., 2003). One of the objectives of the gene expression analysis was to delineate between those genes that underlie the muscle hypertrophy in Callipyge sheep and those that result from the fibre type change in the affected muscle. To address this issue a comparison was undertaken of gene expression in wild type skeletal muscles with differing fibre type compositions to identify fibre type specific genes. These samples were taken from NN animals at T12. The three skeletal muscles used in this analysis were semimembranosis (SM), semitendinosis (ST) and longissimus dorsi (LD).
Project description:In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between cytoplasm and nuclei, and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific transcription and splicing of various sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.