National Center for Health Statistics' 2019 Research and Development Survey, RANDS 3.

Objective This report on the third round of the Research and Development Survey (RANDS 3) provides a general description of RANDS 3 and presents percentage estimates of selected demographic and health-related variables from the overall sample and by one set of experimental groups embedded in the survey. Statistical tests comparing estimates for the two randomized groups were conducted to evaluate the randomization. Methods NORC at the University of Chicago conducted RANDS 3 for the National Center of Health Statistics in 2019 using its AmeriSpeak Panel in web-only mode. To assess question-response patterns, probe questions and four sets of experiments were embedded in RANDS 3, with panelists randomized into two groups for each set of experiments. Participants in each group received questions with differences in wording, question-andresponse formats, or question order. Results Of the 4,255 people sampled, 2,646 completed RANDS 3 for a completion rate of 62.2% and a weighted cumulative response rate of 18.1%. Iterative raking was performed using demographic and selected health condition variables to calibrate the RANDS 3 sample to 2019 National Health Interview Survey (NHIS) estimates. As a result, the overall demographic distribution and percentages of asthma, diabetes, hypertension, and high cholesterol for the calibrated RANDS 3 sample aligned with the percentages estimated from the 2019 NHIS. The distributions of demographic and healthrelated variables were comparable between the two randomized groups examined except for ever-diagnosed hypertension. Conclusion As part of a research series using probability-based survey panels, RANDS 3 included health-related questions with a focus on disability and opioids. Because RANDS is an ongoing research platform, a variety of persistent and emergent research questions relating to survey methodology will continue to be examined in current and future rounds of RANDS.

Proteomic Analysis of Myocardia Containing the Obscurin R4344Q Mutation Linked to Hypertrophic Cardiomyopathy.

Obscurin is a giant cytoskeletal protein with structural and regulatory roles encoded by the OBSCN gene. Recently, mutations in OBSCN were associated with the development of different forms of cardiomyopathies, including hypertrophic cardiomyopathy (HCM). We previously reported that homozygous mice carrying the HCM-linked R4344Q obscurin mutation develop arrhythmia by 1-year of age under sedentary conditions characterized by increased heart rate, frequent incidents of premature ventricular contractions, and episodes of spontaneous ventricular tachycardia. In an effort to delineate the molecular mechanisms that contribute to the observed arrhythmic phenotype, we subjected protein lysates prepared from left ventricles of 1-year old R4344Q and wild-type mice to comparative proteomics analysis using tandem mass spectrometry; raw data are available via ProteomeXchange with identifier PXD017314. We found that the expression levels of proteins involved in cardiac function and disease, cytoskeletal organization, electropotential regulation, molecular transport and metabolism were significantly altered. Moreover, phospho-proteomic evaluation revealed changes in the phosphorylation profile of Ca2+ cycling proteins, including sAnk1.5, a major binding partner of obscurin localized in the sarcoplasmic reticulum; notably, this is the first report indicating that sAnk1 undergoes phosphorylation. Taken together, our findings implicate obscurin in diverse cellular processes within the myocardium, which is consistent with its multiple binding partners, localization in different subcellular compartments, and disease association.

Thick Filament Protein Network, Functions, and Disease Association.

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018.

Correction: Obscurins: Goliaths and Davids Take over Non-Muscle Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0088162.].

Deregulated Ca2+ cycling underlies the development of arrhythmia and heart disease due to mutant obscurin.

Obscurins are cytoskeletal proteins with structural and regulatory roles encoded by OBSCN. Mutations in OBSCN are associated with the development of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Specifically, the R4344Q mutation present in immunoglobulin domain 58 (Ig58) was the first to be linked with the development of HCM. To assess the effects of R4344Q in vivo, we generated the respective knock-in mouse model. Mutant obscurins are expressed and incorporated normally into sarcomeres. The expression patterns of sarcomeric and Ca2+-cycling proteins are unaltered in sedentary 1-year-old knock-in myocardia, with the exception of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase 2 (SERCA2) and pentameric phospholamban whose levels are significantly increased and decreased, respectively. Isolated cardiomyocytes from 1-year-old knock-in hearts exhibit increased Ca2+-transients and Ca2+-load in the sarcoplasmic reticulum and faster contractility kinetics. Moreover, sedentary 1-year-old knock-in animals develop tachycardia accompanied by premature ventricular contractions, whereas 2-month-old knock-in animals subjected to pressure overload develop a DCM-like phenotype. Structural analysis revealed that the R4344Q mutation alters the distribution of electrostatic charges over the Ig58 surface, thus interfering with its binding capabilities. Consistent with this, wild-type Ig58 interacts with phospholamban modestly, and this interaction is markedly enhanced in the presence of R4344Q. Together, our studies demonstrate that under sedentary conditions, the R4344Q mutation results in Ca2+ deregulation and spontaneous arrhythmia, whereas in the presence of chronic, pathological stress, it leads to cardiac remodeling and dilation. We postulate that enhanced binding between mutant obscurins and phospholamban leads to SERCA2 disinhibition, which may underlie the observed pathological alterations.

The sarcomeric M-region: a molecular command center for diverse cellular processes.

The sarcomeric M-region anchors thick filaments and withstands the mechanical stress of contractions by deformation, thus enabling distribution of physiological forces along the length of thick filaments. While the role of the M-region in supporting myofibrillar structure and contractility is well established, its role in mediating additional cellular processes has only recently started to emerge. As such, M-region is the hub of key protein players contributing to cytoskeletal remodeling, signal transduction, mechanosensing, metabolism, and proteasomal degradation. Mutations in genes encoding M-region related proteins lead to development of severe and lethal cardiac and skeletal myopathies affecting mankind. Herein, we describe the main cellular processes taking place at the M-region, other than thick filament assembly, and discuss human myopathies associated with mutant or truncated M-region proteins.

Obscurins: Goliaths and Davids take over non-muscle tissues.

Obscurins comprise a family of proteins originally identified in striated muscles, where they play essential roles in myofibrillogenesis, cytoskeletal organization, and Ca(2+) homeostasis. They are encoded by the single OBSCN gene, and are composed of tandem adhesion domains and signaling motifs. To date, two giant obscurin isoforms have been described in detail that differ only at the extreme COOH-terminus; while obscurin-A (∼720 kDa) contains a non-modular COOH-terminus that harbors binding sites for the adaptor proteins ankyrins, obscurin-B (∼870 kDa) contains two COOH-terminal serine-threonine kinase domains preceded by adhesion motifs. Besides the two known giant obscurins, a thorough search of transcript databases suggests that complex alternative splicing of the obscurin transcript results in the generation of additional giant as well as small isoforms with molecular masses ranging between ∼50-970 kDa. These novel isoforms share common domains with the characterized isoforms, but also contain unique regions. Using a panel of highly specific antibodies directed against epitopes spanning the entire length of giant obscurins, we employed western blotting and immunohistochemistry to perform a systematic and comprehensive characterization of the expression profile of obscurins in muscle and non-muscle tissues. Our studies demonstrate for the first time that obscurins are not restricted to striated muscles, but are abundantly expressed in several tissues and organs including brain, skin, kidney, liver, spleen, and lung. While some obscurin isoforms are ubiquitously expressed, others are preferentially present in specific tissues and organs. Moreover, obscurins are present in select structures and cell types where they assume nuclear, cytosolic, and membrane distributions. Given the ubiquitous expression of some obscurins, along with the preferential expression of others, it becomes apparent that obscurins may play common and unique roles, respectively, in the regulation and maintenance of cell homeostasis in various tissues and organs throughout the body.

Obscurins: unassuming giants enter the spotlight.

Discovered about a decade ago, obscurin (~720 kDa) is a member of a family of giant proteins expressed in striated muscle that are essential for normal muscle function. Much of what we understand about obscurin stems from its functions in cardiac and skeletal muscle. However, recent evidence has indicated that variants of obscurin ("obscurins") are expressed in diverse cell types, where they contribute to distinct cellular processes. Dysfunction or abrogation of obscurins has also been implicated in the development of several pathological conditions, including cardiac hypertrophy and cancer. Herein, we present an overview of obscurins with an emphasis on novel findings that demonstrate their heretofore-unsuspected importance in cell signaling and disease progression.

The kinase domains of obscurin interact with intercellular adhesion proteins.

Obscurins comprise a family of giant (~870- to 600-kDa) and small (~250- to 55-kDa) proteins that play important roles in myofibrillogenesis, cytoskeletal organization, and cell adhesion and are implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including 2 serine/threonine kinase domains, SK1 and SK2, present at the COOH terminus of giant obscurin-B. Using biochemical and cellular approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and undergo autophosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin, a major component of adherens junctions, and SK1 can interact with the extracellular domain of the ß1-subunit of the Na(+)/K(+)-ATPase, which also resides in adherens junctions. Immunostaining of nonpermeabilized myofibers and cardiocytes revealed that some obscurin kinase isoforms localize extracellularly. Quantification of the exofacial expression of obscurin kinase proteins indicated that they occupy ~16 and ~5% of the sarcolemmal surface in myofibers and cardiocytes, respectively. Treatment of heart lysates with peptide-N-glycosidase F revealed that while giant obscurin-B localizes intracellularly, possessing dual kinase activity, a small obscurin kinase isoform that contains SK1 localizes extracellularly, where it undergoes N-glycosylation. Collectively, our studies demonstrate that the obscurin kinase domains are enzymatically active and may be involved in the regulation of cell adhesion.

Obscurin interacts with a novel isoform of MyBP-C slow at the periphery of the sarcomeric M-band and regulates thick filament assembly.

Obscurin is a multidomain protein composed of adhesion and signaling domains that plays key roles in the organization of contractile and membrane structures in striated muscles. Overexpression of the second immunoglobulin domain of obscurin (Ig2) in developing myotubes inhibits the assembly of A- and M-bands, but not Z-disks or I-bands. This effect is mediated by the direct interaction of the Ig2 domain of obscurin with a novel isoform of myosin binding protein-C slow (MyBP-C slow), corresponding to variant-1. Variant-1 contains all the structural motifs present in the known forms of MyBP-C slow, but it has a unique COOH terminus. Quantitative reverse transcription-polymerase chain reaction indicated that MyBP-C slow variant-1 is expressed in skeletal muscles both during development and at maturity. Immunolabeling of skeletal myofibers with antibodies to the unique COOH terminus of variant-1 demonstrated that, unlike other forms of MyBP-C slow that reside in the C-zones of A-bands, variant-1 preferentially concentrates around M-bands, where it codistributes with obscurin. Overexpression of the Ig2 domain of obscurin or reduction of expression of obscurin inhibited the integration of variant-1 into forming M-bands in skeletal myotubes. Collectively, our experiments identify a new ligand of obscurin at the M-band, MyBP-C slow variant-1 and suggest that their interaction contributes to the assembly of M- and A-bands.