Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Strengthening US Food Policies and Programs to Promote Equity in Nutrition Security: A Policy Statement From the American Heart Association.

Nutritionally inadequate dietary intake is a leading contributor to chronic cardiometabolic diseases. Differences in dietary quality contribute to socioeconomic and racial and ethnic health disparities. Food insecurity, a household-level social or economic condition of limited access to sufficient food, is a common cause of inadequate dietary intake. Although US food assistance policies and programs are designed to improve food security, there is growing consensus that they should have a broader focus on nutrition security. In this policy statement, we define nutrition security as an individual or household condition of having equitable and stable availability, access, affordability, and utilization of foods and beverages that promote well-being and prevent and treat disease. Despite existing policies and programs, significant gaps remain for achieving equity in nutrition security across the life span. We provide recommendations for expanding and improving current food assistance policies and programs to achieve nutrition security. These recommendations are guided by several overarching principles: emphasizing nutritional quality, improving reach, ensuring optimal utilization, improving coordination across programs, ensuring stability of access to programs across the life course, and ensuring equity and dignity for access and utilization. We suggest a critical next step will be to develop and implement national measures of nutrition security that can be added to the current US food security measures. Achieving equity in nutrition security will require coordinated and sustained efforts at the federal, state, and local levels. Future advocacy, innovation, and research will be needed to expand existing food assistance policies and programs and to develop and implement new policies and programs that will improve cardiovascular health and reduce disparities in chronic disease.

Chronic Diclofenac Exposure Increases Mitochondrial Oxidative Stress, Inflammatory Mediators, and Cardiac Dysfunction.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly prescribed medications for pain and inflammation. Diclofenac (DIC) is a commonly prescribed NSAID that is known to increase the risk of cardiovascular diseases. However, the mechanisms underlying its cardiotoxic effects remain largely unknown. In this study, we tested the hypothesis that chronic exposure to DIC increases oxidative stress, which ultimately impairs cardiovascular function. METHODS AND RESULTS: Mice were treated with DIC for 4 weeks and subsequently subjected to in vivo and in vitro functional assessments. Chronic DIC exposure resulted in not only systolic but also diastolic dysfunction. DIC treatment, however, did not alter blood pressure or electrocardiographic recordings. Importantly, treatment with DIC significantly increased inflammatory cytokines and chemokines as well as cardiac fibroblast activation and proliferation. There was increased reactive oxygen species (ROS) production in cardiomyocytes from DIC-treated mice, which may contribute to the more depolarized mitochondrial membrane potential and reduced energy production, leading to a significant decrease in sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transients, and sarcomere shortening. Using unbiased metabolomic analyses, we demonstrated significant alterations in oxylipin profiles towards inflammatory features in chronic DIC treatment. CONCLUSIONS: Together, chronic treatment with DIC resulted in severe cardiotoxicity, which was mediated, in part, by an increase in mitochondrial oxidative stress.

Mitochondrial and Proteasome Dysfunction Occurs in the Hearts of Mice Treated with Triazine Herbicide Prometryn.

Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.

A Ketogenic Diet in Combination with Gemcitabine Mitigates Pancreatic Cancer-Associated Cachexia in Male and Female KPC Mice.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

Sarcomeric perturbations of myosin motors lead to dilated cardiomyopathy in genetically modified MYL2 mice.

Dilated cardiomyopathy (DCM) is a devastating heart disease that affects about 1 million people in the United States, but the underlying mechanisms remain poorly understood. In this study, we aimed to determine the biomechanical and structural causes of DCM in transgenic mice carrying a novel mutation in the MYL2 gene, encoding the cardiac myosin regulatory light chain. Transgenic D94A (aspartic acid-to-alanine) mice were created and investigated by echocardiography and invasive hemodynamic and molecular structural and functional assessments. Consistent with the DCM phenotype, a significant reduction of the ejection fraction (EF) was observed in ∼5- and ∼12-mo-old male and female D94A lines compared with respective WT controls. Younger male D94A mice showed a more pronounced left ventricular (LV) chamber dilation compared with female counterparts, but both sexes of D94A lines developed DCM by 12 mo of age. The hypocontractile activity of D94A myosin motors resulted in the rightward shift of the force-pCa dependence and decreased actin-activated myosin ATPase activity. Consistent with a decreased Ca2+ sensitivity of contractile force, a small-angle X-ray diffraction study, performed in D94A fibers at submaximal Ca2+ concentrations, revealed repositioning of the D94A cross-bridge mass toward the thick-filament backbone supporting the hypocontractile state of D94A myosin motors. Our data suggest that structural perturbations at the level of sarcomeres result in aberrant cardiomyocyte cytoarchitecture and lead to LV chamber dilation and decreased EF, manifesting in systolic dysfunction of D94A hearts. The D94A-induced development of DCM in mice closely follows the clinical phenotype and suggests that MYL2 may serve as a new therapeutic target for dilated cardiomyopathy.

Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain.

Myosin light chain 2 ( MYL2) gene encodes the myosin regulatory light chain (RLC) simultaneously in heart ventricles and in slow-twitch skeletal muscle. Using transgenic mice with cardiac-specific expression of the human R58Q-RLC mutant, we sought to determine whether the hypertrophic cardiomyopathy phenotype observed in papillary muscles (PMs) of R58Q mice is also manifested in slow-twitch soleus (SOL) muscles. Skinned SOL muscles and ventricular PMs of R58Q animals exhibited lower contractile force that was not observed in the fast-twitch extensor digitorum longus muscles of R58Q vs. wild-type-RLC mice, but mutant animals did not display gross muscle weakness in vivo. Consistent with SOL muscle abnormalities in R58Q vs. wild-type mice, myosin ATPase staining revealed a decreased proportion of fiber type I/type II only in SOL muscles but not in the extensor digitorum longus muscles. The similarities between SOL muscles and PMs of R58Q mice were further supported by quantitative proteomics. Differential regulation of proteins involved in energy metabolism, cell-cell interactions, and protein-protein signaling was concurrently observed in the hearts and SOL muscles of R58Q mice. In summary, even though R58Q expression was restricted to the heart of mice, functional similarities were clearly observed between the hearts and slow-twitch skeletal muscle, suggesting that MYL2 mutated models of hypertrophic cardiomyopathy may be useful research tools to study the molecular, structural, and energetic mechanisms of cardioskeletal myopathy associated with myosin RLC.-Kazmierczak, K., Liang, J., Yuan, C.-C., Yadav, S., Sitbon, Y. H., Walz, K., Ma, W., Irving, T. C., Cheah, J. X., Gomes, A. V., Szczesna-Cordary, D. Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain.

Impaired proteostasis in senescent vascular endothelial cells: a perspective on estrogen and oxidative stress in the aging vasculature.

The heat shock response is an important cytoprotective mechanism for protein homeostasis and is an essential protective response to cellular stress and injury. Studies on changes in the heat shock response with aging have been mixed with regard to whether it is inhibited, and this, at least in part, reflects different tissues and different models. Cellular senescence is a key feature in aging, but work on the heat shock response in cultured senescent (SEN) cells has largely been limited to fibroblasts. Given the prevalence of oxidative injury in the aging cardiovascular system, we investigated whether SEN primary human coronary artery endothelial cells have a diminished heat shock response and impaired proteostasis. In addition, we tested whether this downregulation of heat shock response can be mitigated by 17ß-estradiol (E2), which has a critical cardioprotective role in women, as we have previously reported that E2 improves the heat shock response in endothelial cells (Hamilton KL, Mbai FN, Gupta S, Knowlton AA. Arterioscler Thromb Vasc Biol 24: 1628-1633, 2004). We found that SEN endothelial cells, despite their unexpectedly increased proteasome activity, had a diminished heat shock response and had more protein aggregation than early passage cells. SEN cells had increased oxidative stress, which promoted protein aggregation. E2 treatment did not decrease protein aggregation or improve the heat shock response in either early passage or SEN cells. In summary, cellular senescence in adult human endothelial cells is accompanied by increased oxidative stress and a blunting of proteostasis, and E2 did not mitigate these changes. NEW & NOTEWORTHY Senescent human endothelial cells have a diminished heat shock response and increased protein aggregates. Senescent human endothelial cells have increased basal oxidative stress, which increases protein aggregates. Physiological level of 17ß-estradiol did not improve proteostasis in endothelial cells.

Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP).

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 µg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.

Ponceau S waste: Ponceau S staining for total protein normalization.

Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 and 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used.

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations.

Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.

Proteasome dysfunction in cardiomyopathies.

The ubiquitin-proteasome system (UPS) plays a critical role in removing unwanted intracellular proteins and is involved in protein quality control, signalling and cell death. Because the heart is subject to continuous metabolic and mechanical stress, the proteasome plays a particularly important role in the heart, and proteasome dysfunction has been suggested as a causative factor in cardiac dysfunction. Proteasome impairment has been detected in cardiomyopathies, heart failure, myocardial ischaemia, and hypertrophy. Proteasome inhibition is also sufficient to cause cardiac dysfunction in healthy pigs, and patients using a proteasome inhibitor for cancer therapy have a higher incidence of heart failure. In this Topical Review we discuss the experimental data which suggest UPS dysfunction is a common feature of cardiomyopathies, with an emphasis on hypertrophic cardiomyopathy caused by sarcomeric mutations. We also propose potential mechanisms by which cardiomyopathy-causing mutations may lead to proteasome impairment, such as altered calcium handling and increased oxidative stress due to mitochondrial dysfunction.

Protein purification and analysis: next generation Western blotting techniques.

INTRODUCTION: Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

Modulation of mitochondrial dysfunction and endoplasmic reticulum stress are key mechanisms for the wide-ranging actions of epoxy fatty acids and soluble epoxide hydrolase inhibitors.

The arachidonic acid cascade is arguably the most widely known biologic regulatory pathway. Decades after the seminal discoveries involving its cyclooxygenase and lipoxygenase branches, studies of this cascade remain an active area of research. The third and less widely known branch, the cytochrome P450 pathway leads to highly active oxygenated lipid mediators, epoxy fatty acids (EpFAs) and hydroxyeicosatetraenoic acids (HETEs), which are of similar potency to prostanoids and leukotrienes. Unlike the COX and LOX branches, no pharmaceuticals currently are marketed targeting the P450 branch. However, data support therapeutic benefits from modulating these regulatory lipid mediators. This is being approached by stabilizing or mimicking the EpFAs or even by altering the diet. These approaches lead to predominantly beneficial effects on a wide range of apparently unrelated states resulting in an enigma of how this small group of natural chemical mediators can have such diverse effects. EpFAs are degraded by soluble epoxide hydrolase (sEH) and stabilized by inhibiting this enzyme. In this review, we focus on interconnected aspects of reported mechanisms of action of EpFAs and inhibitors of soluble epoxide hydrolase (sEHI). The sEHI and EpFAs are commonly reported to maintain homeostasis under pathological conditions while remaining neutral under normal physiological conditions. Here we provide a conceptual framework for the unique and broad range of biological activities ascribed to epoxy fatty acids. We argue that their mechanism of action pivots on their ability to prevent mitochondrial dysfunction, to reduce subsequent ROS formation and to block resulting cellular signaling cascades, primarily the endoplasmic reticulum stress. By stabilizing the mitochondrial - ROS - ER stress axis, the range of activity of EpFAs and sEHI display an overlap with the disease conditions including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative diseases, for which the above outlined mechanisms play key roles.

Different effects of the nonsteroidal anti-inflammatory drugs meclofenamate sodium and naproxen sodium on proteasome activity in cardiac cells.

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods.

Acute resistance exercise activates rapamycin-sensitive and -insensitive mechanisms that control translational activity and capacity in skeletal muscle.

KEY POINTS: Ribosome biogenesis is the primary determinant of translational capacity, but its regulation in skeletal muscle following acute resistance exercise is poorly understood. Resistance exercise increases muscle protein synthesis acutely, and muscle mass with training, but the role of translational capacity in these processes is unclear. Here, we show that acute resistance exercise activated pathways controlling translational activity and capacity through both rapamycin-sensitive and -insensitive mechanisms. Transcription factor c-Myc and its downstream targets, which are known to regulate ribosome biogenesis in other cell types, were upregulated after resistance exercise in a rapamycin-independent manner and may play a role in determining translational capacity in skeletal muscle. Local inhibition of myostatin was also not affected by rapamycin and may contribute to the rapamycin-independent effects of resistance exercise. ABSTRACT: This study aimed to determine (1) the effect of acute resistance exercise on mechanisms of ribosome biogenesis, and (2) the impact of mammalian target of rapamycin on ribosome biogenesis, and muscle protein synthesis (MPS) and degradation. Female F344BN rats underwent unilateral electrical stimulation of the sciatic nerve to mimic resistance exercise in the tibialis anterior (TA) muscle. TA muscles were collected at intervals over the 36 h of exercise recovery (REx); separate groups of animals were administered rapamycin pre-exercise (REx+Rapamycin). Resistance exercise led to a prolonged (6-36 h) elevation (30-50%) of MPS that was fully blocked by rapamycin at 6 h but only partially at 18 h. REx also altered pathways that regulate protein homeostasis and mRNA translation in a manner that was both rapamycin-sensitive (proteasome activity; phosphorylation of S6K1 and rpS6) and rapamycin-insensitive (phosphorylation of eEF2, ERK1/2 and UBF; gene expression of the myostatin target Mighty as well as c-Myc and its targets involved in ribosome biogenesis). The role of c-Myc was tested in vitro using the inhibitor 10058-F4, which, over time, decreased basal RNA and MPS in a dose-dependent manner (correlation of RNA and MPS, r(2) = 0.98), even though it had no effect on the acute stimulation of protein synthesis. In conclusion, acute resistance exercise stimulated rapamycin-sensitive and -insensitive mechanisms that regulate translation activity and capacity.

Transformative Impact of Proteomics on Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.

The year 2014 marked the 20th anniversary of the coining of the term proteomics. The purpose of this scientific statement is to summarize advances over this period that have catalyzed our capacity to address the experimental, translational, and clinical implications of proteomics as applied to cardiovascular health and disease and to evaluate the current status of the field. Key successes that have energized the field are delineated; opportunities for proteomics to drive basic science research, facilitate clinical translation, and establish diagnostic and therapeutic healthcare algorithms are discussed; and challenges that remain to be solved before proteomic technologies can be readily translated from scientific discoveries to meaningful advances in cardiovascular care are addressed. Proteomics is the result of disruptive technologies, namely, mass spectrometry and database searching, which drove protein analysis from 1 protein at a time to protein mixture analyses that enable large-scale analysis of proteins and facilitate paradigm shifts in biological concepts that address important clinical questions. Over the past 20 years, the field of proteomics has matured, yet it is still developing rapidly. The scope of this statement will extend beyond the reaches of a typical review article and offer guidance on the use of next-generation proteomics for future scientific discovery in the basic research laboratory and clinical settings.

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice.

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.