Expression Of Sarcomeric Proteins Research Articles

Abstract P1025: Hsp70 Co-chaperones Regulate Sarcomeric Proteostasis In Hypertrophic Cardiomyopathy

Significance: Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric variants in hypertrophic cardiomyopathy (HCM) with three of the top 10 variant loci in or near genes that encode for co-chaperones of HSP70: BAG3, DNAJC18 and HSPB7. These variants are also present in dilated cardiomyopathy (DCM), but have an opposing effect on cardiac function, leading us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) had on sarcomeric and z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins. Methods and Results: Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies > 75% and >80% reduction in target transcript 4 days post-transduction. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of sarcomeric and z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomere protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). DNAJC18 KD increased BAG3 and HSPB7 protein expression levels by ~2-fold (p<0.001) without increasing transcript levels, suggesting BAG3 and HSPB7 may be protein clients of DNAJC18. Conclusions: HSP70 co-chaperones that have an established genetic association with human HCM play an important role in cardiomyocyte proteostasis. Multiple sarcomeric and z-disc proteins appear to be clients of BAG3, while BAG3 and HSPB7 themselves may be clients of DNAJC18. Targeting this axis could be therapeutic for patients with HCM that possess genetic variants in sarcomeric proteins.

Targeting co-chaperones of HSP70 to modulate Myosin Binding Protein C turnover in hypertrophic cardiomyopathy.

Rationale: Hypertrophic Cardiomyopathy (HCM) is an inherited cardiovascular disease that affects 1 in 500 people. Over half of familial HCM cases arise from MyBP-C haploinsufficiency, thus stabilizing MyBP-C protein expression is an appealing therapeutic approach. Recently, two GWAS studies identified multiple HCM risk alleles. Three of the top 10 are localized near or in gene loci that code for HSP70 co-chaperones, highlighting the important role of the HSP70 system in HCM. Hypothesis: Co-chaperones of HSP70, (BAG3, DNAJC18, and HSPB7) regulate the expression and stability of sarcomeric and z-disc proteins, specifically MyBP-C. Methods/Results: Proximity labeling was performed to identify which components of the HSP70 co-chaperone network interact with or lie in close proximity to MyBP-C. Human iPSC-CMs (hiPSC-CMs) were transduced with a Turbo-ID-MYBPC3 adenoviral (AdV) construct, treated with biotin, and biotinylated proteins were captured by streptavidin beads. HSPA1A (i.e. HSP70), BAG3, DNAJC13, HSPB7, HSPB1, and CRYAB were identified by mass spectrometry as proximity proteins to wild-type MyBP-C but were absent in controls (AdV-MyBP-C without Turbo). hiPSC-CMs were transfected with AdV-shRNA to knock down HSP70 co-chaperone expression (BAG3, DNAJC18, or HSPB7). DNAJC18 was included because of the risk allele identified in GWAS, even though DNAJC18 was not identified as a direct interactor with MyBP-C during proximity labeling. Adv transfection efficiency was > 70% (p<0.0001) and resulted in a >75% decrease (p<0.01) in co-chaperone transcript expression and a >50% decrease (p<0.001) in protein. BAG3 knockdown decreased the steady state protein expression of MyBP-C (p<0.0001) and a majority of other sarcomeric proteins. Utilizing a cycloheximide (CHX) chase assay to measure protein ½ life, MyBP-C protein degradation was greatly accelerated by BAG3 knockdown compared to scrambled shRNA 24 hours post-CHX (p<0.0001). DNAJC18 knockdown had no significant effects on steady state protein expression of MyBP-C or other sarcomeric proteins, while HSPB7 knockdown increased MyBP-C (p<0.01), myosin (p<0.01), tropomyosin (p<0.05), and myosin light chain 2 (p<0.05) protein compared to scrambled shRNA. DNAJC18 and HSPB7 knockdown each increased BAG3 protein expression (p<0.01), while BAG3 knockdown reduced DNAJC18 protein (p<0.0001) but had no effect on HSPB7. Conclusion: MyBP-C interacts with and is stabilized by BAG3. MyBP-C may also be a direct client for HSPB7, but further studies are needed. Knockdown experiments of DNAJC13, HSBP1 and CRYAB, identified as proximity proteins of MyBP-C by Turbo-ID, are also underway. The HSP70 co-chaperone network may serve a therapeutic target to normalize MyBP-C levels in the context of MyBP-C haploinsufficiency. Institutional Start Up Funds This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vascular cells improve functionality of human cardiac organoids

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models.

USP18 is an essential regulator of muscle cell differentiation and maturation

The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response.

Embryonic Hyperglycemia Disrupts Myocardial Growth, Morphological Development, and Cellular Organization: An In Vivo Experimental Study.

Hyperglycemia during gestation can disrupt fetal heart development and increase postnatal cardiovascular disease risk. It is therefore imperative to identify early biomarkers of hyperglycemia during gestation-induced fetal heart damage and elucidate the underlying molecular pathomechanisms. Clinical investigations of diabetic adults with heart dysfunction and transgenic mouse studies have revealed that overexpression or increased expression of TNNI3K, a heart-specific kinase that binds troponin cardiac I, may contribute to abnormal cardiac remodeling, ventricular hypertrophy, and heart failure. Optimal heart function also depends on the precise organization of contractile and excitable tissues conferred by intercellular occlusive, adherent, and communicating junctions. The current study evaluated changes in embryonic heart development and the expression levels of sarcomeric proteins (troponin I, desmin, and TNNI3K), junctional proteins, glucose transporter-1, and Ki-67 under fetal hyperglycemia. Stage 22HH Gallus domesticus embryos were randomly divided into two groups: a hyperglycemia (HG) group, in which individual embryos were injected with 30 mmol/L glucose solution every 24 h for 10 days, and a no-treatment (NT) control group, in which individual embryos were injected with physiological saline every 24 h for 10 days (stage 36HH). Embryonic blood glucose, height, and weight, as well as heart size, were measured periodically during treatment, followed by histopathological analysis and estimation of sarcomeric and junctional protein expression by western blotting and immunostaining. Hyperglycemic embryos demonstrated delayed heart maturation, with histopathological analysis revealing reduced left and right ventricular wall thickness (-39% and -35% vs. NT). Immunoexpression levels of TNNI3K and troponin 1 increased (by 37% and 39%, respectively), and desmin immunofluorescence reduced (by 23%). Embryo-fetal hyperglycemia may trigger an increase in the expression levels of TNNI3K and troponin I, as well as dysfunction of occlusive and adherent junctions, ultimately inducing abnormal cardiac remodeling.

Sodium fluoride induces skeletal muscle atrophy via changes in mitochondrial and sarcomeric proteomes.

Sodium Fluoride (NaF) can change the expression of skeletal muscle proteins. Since skeletal muscle is rich in mitochondrial and contractile (sarcomeric) proteins, these proteins are sensitive to the effects of NaF, and the changes are dose-and time-dependent. In the current study, we have analysed the effect of high concentrations of NaF (80ppm) on mouse skeletal muscle at two different time points, i.e., 15 days and 60 days. At the end of the experimental time, the animals were sacrificed, skeletal muscles were isolated, and proteins were extracted and subjected to bioinformatic (Mass Spectrometric) analysis. The results were analysed based on changes in different mitochondrial complexes, contractile (sarcomeric) proteins, 26S proteasome, and ubiquitin-proteasome pathway. The results showed that the mitochondrial proteins of complex I, II, III, IV and V were differentially regulated in the groups treated with 80ppm of NaF for 15 days and 60 days. The network analysis indicated more changes in mitochondrial proteins in the group treated with the higher dose for 15 days rather than 60 days. Furthermore, differential expression of (sarcomeric) proteins, downregulation of 26S proteasome subunits, and differential expression in proteins related to the ubiquitin-proteasome pathway lead to muscle atrophy. The differential expression might be due to the adaptative mechanism to counteract the deleterious effects of NaF on energy metabolism. Data are available via ProteomeXchange with identifier PXD035014.

Switching in the expression pattern of actin isoforms marks the onset of contractility and distinct mechanodynamic behavior during cardiomyocyte differentiation.

Differentiation of cardiac progenitor cells (CPC) into cardiomyocytes is a fundamental step in cardiogenesis, which is marked by changes in gene expression responsible for remodeling of the cytoskeleton and in altering the mechanical properties of cells. Here we have induced the differentiation of CPC derived from human pluripotent stem cells into immature cardiomyocytes (iCM) which we compare with more differentiated cardiomyocytes (mCM). Using atomic force microscopy and real‐time deformability cytometry, we describe the mechanodynamic changes that occur during the differentiation process and link our findings to protein expression data of cytoskeletal proteins. Increased levels of cardiac‐specific markers as well as evolution of cytoskeletal morphology and contractility parameters correlated with the expected extent of cell differentiation that was accompanied by hypertrophic growth of cells. These changes were associated with switching in the balance of the different actin isoforms where β‐actin is predominantly found in CPC, smooth muscle α‐actin is dominant in iCM cells and sarcomeric α‐actin is found in significantly higher levels in mCM. We link these cytoskeletal changes to differences in mechano‐dynamic behavior of cells that translate to changes in Young's modulus that depend on the cell adherence. Our results demonstrate that the intracellular balance of actin isoform expression can be used as a sensitive ruler to determine the stage of differentiation during early phases of cardiomyocyte differentiation that correlates with an increased expression of sarcomeric proteins and is accompanied by changes in cellular elasticity.

Development of a Vascularised Cardiac Organoid Platform Reveals a Regulatory Role for Extracellular Matrix Environment on Muscle Functionality

Multicellular composition of the heart is important for regulating organ development and responses to injury, however the role of cardiac cell types in organ maturation is not well characterised. In this study we characterised a vascularised cardiac organoid model (vhCO) derived from human pluripotent stem cells to better understand cell-cell interactions in human heart tissue. We show that endothelial cells in vhCO increase organoid force of contraction and enhance the expression of mature sarcomeric proteins and extracellular matrix (ECM) components.

Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle

Complex animals build specialised muscles to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres and mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we use Drosophila muscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selector spalt instructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated leg muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this causes the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream of spalt synchronizing mitochondria with myofibril morphogenesis.

Loss of sarcomeric proteins via upregulation of JAK/STAT signaling underlies interferon-γ-induced contractile deficit in engineered human myocardium

The level of circulating interferon-γ (IFNγ) is elevated in various clinical conditions including autoimmune and inflammatory diseases, sepsis, acute coronary syndrome, and viral infections. As these conditions are associated with high risk of myocardial dysfunction, we investigated the effects of IFNγ on 3D fibrin-based engineered human cardiac tissues (“cardiobundles”). Cardiobundles were fabricated from human pluripotent stem cell-derived cardiomyocytes, exposed to 0–20 ng/ml of IFNγ on culture days 7–14, and assessed for changes in tissue structure, viability, contractile force and calcium transient generation, action potential propagation, cytokine secretion, and expression of select genes and proteins. We found that application of IFNγ induced a dose-dependent reduction in contractile force generation, deterioration of sarcomeric organization, and cardiomyocyte disarray, without significantly altering cell viability, action potential propagation, or calcium transient amplitude. At molecular level, the IFNγ-induced structural and functional deficits could be attributed to altered balance of pro- and anti-inflammatory cytokines, upregulation of JAK/STAT signaling pathway (JAK1, JAK2, and STAT1), and reduced expression of myosin heavy chain, myosin light chain-2v, and sarcomeric α-actinin. Application of clinically used JAK/STAT inhibitors, tofacitinib and baricitinib, fully prevented IFNγ-induced cardiomyopathy, confirming the critical roles of this signaling pathway in inflammatory cardiac disease. Taken together, our in vitro studies in engineered myocardial tissues reveal direct adverse effects of pro-inflammatory cytokine IFNγ on human cardiomyocytes and establish the foundation for a potential use of cardiobundle platform in modeling of inflammatory myocardial disease and therapy. Statement of significanceVarious inflammatory and autoimmune diseases including rheumatoid arthritis, sepsis, lupus erythematosus, Chagas disease, and others, as well as viral infections including H1N1 influenza and COVID-19 show increased systemic levels of a pro-inflammatory cytokine interferon-γ (IFNγ) and are associated with high risk of heart disease. Here we explored for the first time if chronically elevated levels of IFNγ can negatively affect structure and function of engineered human heart tissues in vitro. Our studies revealed IFNγ-induced deterioration of myofibrillar organization and contractile force production in human cardiomyocytes, attributed to decreased expression of multiple sarcomeric proteins and upregulation of JAK/STAT signaling pathway. FDA-approved JAK inhibitors fully blocked the adverse effects of IFNγ, suggesting a potentially effective strategy against human inflammatory cardiomyopathy.

Human induced pluripotent stem cell-based platform for modeling cardiac ischemia

Ischemic heart disease is a major cause of death worldwide, and the only available therapy to salvage the tissue is reperfusion, which can initially cause further damage. Many therapeutics that have been promising in animal models have failed in human trials. Thus, functional human based cardiac ischemia models are required. In this study, a human induced pluripotent stem cell derived-cardiomyocyte (hiPSC-CM)-based platform for modeling ischemia–reperfusion was developed utilizing a system enabling precise control over oxygen concentration and real-time monitoring of the oxygen dynamics as well as iPS-CM functionality. In addition, morphology and expression of hypoxia-related genes and proteins were evaluated as hiPSC-CM response to 8 or 24 h hypoxia and 24 h reoxygenation. During hypoxia, initial decrease in hiPSC-CM beating frequency was observed, after which the CMs adapted to the conditions and the beating frequency gradually increased already before reoxygenation. During reoxygenation, the beating frequency typically first surpassed the baseline before settling down to the values close the baseline. Furthermore, slowing on the field potential propagation throughout the hiPSC-CM sheet as well as increase in depolarization time and decrease in overall field potential duration were observed during hypoxia. These changes were reversed during reoxygenation. Disorganization of sarcomere structures was observed after hypoxia and reoxygenation, supported by decrease in the expression of sarcomeric proteins. Furthermore, increase in the expression of gene encoding glucose transporter 1 was observed. These findings indicate, that despite their immature phenotype, hiPSC-CMs can be utilized in modeling ischemia–reperfusion injury.

ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

Cytoskeletal disorganization underlies PABPN1-mediated myogenic disability

Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy.

Abstract 103: TBX20 Activates Cardiac Maturation Gene Programs Promoting Direct Human Cardiac Reprogramming

Direct cardiac reprogramming to generate induced cardiomyocyte like cells (iCMs) from fibroblasts has emerged as a promising therapeutic strategy for the treatment of heart failure, which is still the leading cause of mortality and morbidity in developed countries. While much is known regarding iCMs generated from mouse cells, the adaptation of direct cardiac reprogramming to human cells is hurdled with low efficiency and poor quality because of intrinsic differences between species. Recently, our single cell transcriptomic analysis during human direct cardiac reprogramming demonstrated the immature features of human iCMs (hiCMs) compared to endogenous cardiomyocytes (CMs) at the transcriptome level, suggesting that key factors required for CM maturation remain unidentified. We then utilized computational analyses of genomic and epigenomic data to identify the regulators underexpressed in hiCMs comparing to CMs derived from human heart tissue or stem cell differentiation. Among them, the top hit is T-box family transcription factor TBX20, whose role in direct cardiac reprogramming remains to be explored. Supplementing TBX20 in reprogramming cocktails, we found significantly increased sarcomeric protein expression, like αMHC and αActinin in hiCMs and the formation of well-organized myofibrils. TBX20’s effect has also been supported by transcriptomic analysis, where we found TBX20 activates a large number of underexpressed genes associated with sarcomere structure and ion channels, such as MYH7 , MYL2 , MYBPC3, SCN5A and KCNQ1 , suggesting that TBX20 likely enhances maturation and contractility of hiCMs. We also found that TBX20 cannot replace any of the reprogramming factors, suggesting that its potential role is complimentary to and interacting with the current cocktail. Moreover, known TBX20 targets showed inconsistent alteration in TBX20-transduced hiCMs, suggesting its novel regulation in a direct reprogramming setting. These findings assign a novel reprogramming factor TBX20 playing an essential role in generating mature hiCMs by establishing gene programs associated with cardiac muscle contractility.

A Data-Driven Methodology Reveals Novel Myofiber Clusters in Older Human Muscles

Skeletal muscles control posture, mobility and strength, affecting whole-body metabolism. The muscle is composed of different types of myofibers, which are defined by their contractile properties, and are marked by the expression of sarcomeric proteins. Three myosin heavy-chain (MyHC) isoforms, MyHC-1, MyHC-2A and MyHC-2X characterize myofiber properties in humans, yet characterization of myofibers is often performed considering only a single MyHC isoform expression. We aimed to study heterogeneity in muscle tissue composition, by detailed analysis of the expression of the three MyHC isoforms in consortium. We created a dataset with high-throughput MyHC immunofluorescence imaging in muscle tissues of 56 older adults. Our analyses revealed six distinct myofiber clusters, some of which could not be detected or are misclassified by the traditional visual myofiber assessment. Transcriptome analyses revealed that the abundance of these myofiber clusters was correlated with the expression of distinct modules of sarcomeric genes. Interestingly, one of the novel clusters, expressing all three MyHC isoforms, correlated to multiple histological measures of muscle health. Collectively, we present a data-driven procedure for a deeper characterization of muscle tissue composition, that opens new options to unravel the relations between alterations in muscle composition and changes in a variety of health conditions and aging.

Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome

Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.

Proteomic profiling of skeletal and cardiac muscle in cancer cachexia: alterations in sarcomeric and mitochondrial protein expression.

BackgroundCancer cachexia is observed in more than 50% of advanced cancer patients, and impairs quality of life and prognosis. A variety of pathways are likely to be dysregulated. Hence, a broad-spectrum understanding of the disease process is best achieved by a discovery based approach such as proteomics.ResultsMore than 300 proteins were identified with > 95% confidence in correct sequence identification, of which 5–10% were significantly differentially expressed in cachectic tissues (p-value of 0.05; 27 proteins from gastrocnemius, 34 proteins from soleus and 24 proteins from heart). The two most pronounced functional groups being sarcomeric proteins (mostly upregulated across all three muscle types) and energy/metabolism proteins (mostly downregulated across all muscle types). Electron microscopy revealed disintegration of the sarcomere and morphological aberrations of mitochondria in the cardiac muscle of colon 26 (C26) carcinoma mice.Materials and MethodsThe colon 26 (C26) carcinoma mouse model of cachexia was used to analyse soleus, gastrocnemius and cardiac muscles using two 8-plex iTRAQ proteomic experiments and tandem mass spectrometry (LCMSMS). Differentially expressed proteomic lists for protein clustering and enrichment of biological processes, molecular pathways, and disease related pathways were analysed using bioinformatics. Cardiac muscle ultrastructure was explored by electron microscopy.ConclusionsMorphological and proteomic analyses suggested molecular events associated with disintegrated sarcomeric structure with increased dissolution of Z-disc and M-line proteins. Altered mitochondrial morphology, in combination with the reduced expression of proteins regulating substrate and energy metabolism, suggest that muscle cells are likely to be undergoing a state of energy crisis which ultimately results in cancer-induced cachexia.

Skeletal myofiber VEGF‐A‐targeted gene deletion induces mitochondrial, structural and contractile alterations in mouse diaphragm

Respiratory muscle weakness is a hallmark of COPD, heart failure, and critical illness. Vascular endothelial growth factor (VEGF) may be an important therapeutic target in chronic cardiopulmonary disease as it is essential for neovascularization and vessel repair following trauma. We hypothesized that respiratory muscle, which is required to persistently function, would exhibit impaired mitochondrial, structural, and contractile function in skeletal myofiber VEGF gene deleted mice.We measured diaphragm biochemistry, structure, and contractile function in adult mice with conditional deletion of the VEGF‐A gene in skeletal myofibers (SkmVEGF−/−). Male SkmVEGF−/− mice (n=12) were compared to wild type (WT) controls (n=13) – both on a C57BL/6J background. Fiber bundles were isolated and electrically stimulated in vitro to evaluate force‐frequency and fatigue profiles. Mitochondrial O2 consumption and H2O2 flux (index of mitochondrial ROS) were simultaneously measured in saponin‐permeabilized fiber bundles (Oroboros, Innsbruck, AT). Fiber structure and biochemistry were measured using standard techniques (IHC, Western, ELISA).Diaphragm VEGF protein was lower in SkmVEGF−/− than WT mice (1.5±0.2 vs. 3.0±0.4 pg/total protein, p<0.05). Compared to WT mice, the force‐frequency relationship was depressed in SkmVEGF−/− (F[7,161]=15.0, p<0.05), with maximal specific force reduced by ~10 % (24±1 vs. 21±1 N/cm2; p<0.05). However, fatigue resistance tended to be greater following SkmVEGF−/− (F[2,115]=3.3, p=0.06). Fiber type proportions were unchanged, but type I fiber cross‐sectional area was reduced in SkmVEGF−/− (653±45 vs 493±53 μm2; p<0.05). Compared to WT, sarcomeric actin protein expression was reduced by ~30% in SkmVEGF−/− (p<0.05) while myosin heavy chain, MAFbx, MuRF1, PGC1α, and HIF1α protein remained unchanged (p>0.05). Mitochondrial respiration was not different between SkmVEGF−/− and WT (F[1,80]=0.9, p>0.05) in each of the respiratory states. However, H2O2 flux was reduced in SkmVEGF−/− (F[1,75]=14.6, p<0.05).Skeletal myofiber‐specific VEGF deletion contributed to diaphragm weakness, but tended to improve fatigue resistance in this highly oxidative muscle. Reductions in oxidative fiber size, sarcomeric actin protein content, and ROS generation following VEGF gene deletion may contribute to protecting mitochondrial respiratory function in the diaphragm. The adaptations may be part of compensatory mechanisms mitigating larger deficits in force generation and fatigue resistance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex-Related Differences in Sarcomeric Protein Expression in Guinea Pig Masticatory Muscles

Sex-Related Differences in Sarcomeric Protein Expression in Guinea Pig Masticatory Muscles

HIC2 regulates isoform switching during maturation of the cardiovascular system

Physiological changes during embryonic development are associated with changes in the isoform expression of both myocyte sarcomeric proteins and of erythrocyte haemoglobins. Cell type-specific isoform expression of these genes also occurs. Although these changes appear to be coordinated, it is unclear how changes in these disparate cell types may be linked. The transcription factor Hic2 is required for normal cardiac development and the mutant is embryonic lethal. Hic2 embryos exhibit precocious expression of the definitive-lineage haemoglobin Hbb-bt in circulating primitive erythrocytes and of foetal isoforms of cardiomyocyte genes (creatine kinase, Ckm, and eukaryotic elongation factor Eef1a2) as well as ectopic cardiac expression of fast-twitch skeletal muscle troponin isoforms. We propose that HIC2 regulates a switching event within both the contractile machinery of cardiomyocytes and the oxygen carrying systems during the developmental period where demands on cardiac loading change rapidly.