Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The musclespecific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and ␣-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented Skeletal muscles contain different types of fibers, which are responsible for specific biological properties and functions of individual muscles. Muscle fibers have been classified into slow type I and fast type II fibers mainly based on myofibrillar ATP staining and immunohistochemistry using specific antibodies (1).Slow type I fibers show a red tint, contain high numbers of mitochondria, and their energy supply is mainly based on oxidative metabolism. These features enable slow fibers to execute long lasting contractions, which are essential for the maintenance of body posture. The primary function of type II fibers is the rapid contraction of muscles. Fast fibers are divided into three additional subclasses: Type IIb and IIx (also known as IId) are glycolytic fibers, whereas type IIa fibers are more comparable to oxidative type I fibers (2). Type II fibers, which mainly derive their energy from glycolysis, are thus more susceptible to fatigue compared with Type I fibers.Muscle fibers have also been classified based on the expression of different isoforms of myosin heavy chain (MyHC) proteins. Myosins are the major contractile proteins and their activation by ATP and Ca 2ϩ ions results in shortening of muscle fibers. For example, slow type I fibers express MyHCI␤ a...

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Quantitative proteomics is an important tool to study biological processes, but so far it has been challenging to apply to zebrafish. Here, we describe a large scale quantitative analysis of the zebrafish proteome using a combination of stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS). Proteins derived from the fully labeled fish were used as a standard to quantify changes during embryonic heart development. LC-MS-assisted analysis of the proteome of activated leukocyte cell adhesion molecule zebrafish morphants revealed a down-regulation of components of the network required for cell adhesion and maintenance of cell shape as well as secondary changes due to arrest of cellular differentiation. Quantitative proteomics in zebrafish using the stable isotope-labeling technique provides an unprecedented resource to study developmental processes in zebrafish. Over the past years, mass spectrometry-based proteomics has been widely used to analyze complex biological samples (1). Although the latest generation of MS instrumentation allows proteome-wide analysis, protein quantitation is still a challenge (2, 3). Metabolic labeling using stable isotopes has been used for almost a century. Today, the most commonly used techniques for relative protein quantification are based on 15 N labeling and stable isotope labeling by amino acids in cell culture (SILAC) 1 (4, 5). SILAC was initially developed for cell culture experiments, and recent approaches extended labeling to living organisms, including bacteria (6), yeast (7), flies (8), worms (9), and rodents (10, 11). In addition, several pulsed SILAC (also known as dynamic SILAC) experiments were performed to assess protein dynamics in cell culture and living animals (12-15).The zebrafish (Danio rerio) has proved to be an important model organism to study developmental processes. It also serves as a valuable tool to investigate basic pathogenic principles of human diseases such as cardiovascular disorders and tissue regeneration (16). So far, most researchers rely on immunohistochemistry and Western blots for semiquantitative protein analysis, an approach that is hampered by the paucity of reliable antibodies in zebrafish. Proteomics approaches that depend on two-dimensional gel approaches (17-19) have not gained wide popularity because of issues with workload, reproducibility, and sensitivity (20, 21).Another approach for protein quantitation is the chemical modification of peptides, and so far several isobaric tagging methods, including ICAT (22), iTRAQ (23),18 O (24), and dimethyl labeling (25), have been proven to be successful methods.Recently, a quantitative phosphopeptide study based on dimethyl labeling in zebrafish showed the consequences of a morpholino-based kinase knockdown (26). However, each chemical modification bears the risk of nonspecific and incomplete labeling, which complicates mass spectrometric data interpretation.Alternatively, a metabolic labeling study with stable isotopes was recently performed on adult zebrafish by the admi...

The zebrafish has become a widely used model organism employed for developmental studies, live cell imaging, and genetic screens. High-resolution transcriptional profiles of different developmental and adult stages of the fish and of its various organs were generated, which are readily accessible via the ZFIN database. In contrast, quantitative proteomic studies of zebrafish organs are still in their infancy. Here, we used the SILAC (stable isotope labeling by amino acids in cell culture) zebrafish as a "spike-in" reference to generate a protein atlas of nine organs including gills, brain, heart, muscle, liver, spleen, skin, swim bladder, and testis. Single-shot 4 h LC gradients coupled to a Quadrupole-Orbitrap (QExactive) instrument allowed identification of over 5000 proteins in less than 5 days, of which more than 70% were quantified in triplicate. Identified proteins were subjected to BLAST searches and Gene Ontology classification to improve annotation of zebrafish proteins and obtain insights into potential functions. Comparison to mouse tissue proteome data sets revealed differences and similarities in the protein composition of zebrafish versus mouse organs. We reason that the data set will be helpful for the proteomic characterization of zebrafish organs and identification of tissue-specific proteins that might serve as biomarkers. Our approach provides a complementary view into the biochemistry of zebrafish models and will assist large-scale protein quantification in zebrafish disease models.

Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a (13)C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope (12)C6-lysine. SILAC-labeled organisms are mainly used as a heavy "spike-in" standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed β-parvin-deficient hearts. Our approach confirmed the absence β-parvin and revealed simultaneously a clear up regulation of α-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

Since the first metabolic labeling experiments with stable isotopes beginning of the last century, several approaches were pursued to monitor protein dynamics in living animals. Today, almost all model organisms from bacteria to rodents can be fully labeled with SILAC (stable isotope labeling of amino acids in cell culture) amino acids. The development of special media and diets containing the labeled amino acids provides an efficient way to metabolically label prokaryotic and eukaryotic organisms. Preferentially, the essential amino acid lysine ((13)C6-lysine) is used to label mice (Mus musculus) and after one generation the natural isotope is fully replaced by the stable (13)C6-lysine isotope. So far, the SILAC mouse approach has been used to analyze several transgenic and knockout mouse models. Spike-in of labeled proteins into non-labeled samples provides an accurate relative protein quantification method without any chemical modification. Here we describe how to establish a SILAC mouse colony and describe the analysis of skeletal muscle tissue with different metabolic and contractile profiles.