Assessment of temporal functional changes and miRNA profiling of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation.

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Like R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.

Obscurin is a semi-flexible molecule in solution.

Obscurin, a giant modular cytoskeletal protein, is comprised mostly of tandem immunoglobulin-like (Ig-like) domains. This architecture allows obscurin to connect distal targets within the cell. The linkers connecting the Ig domains are usually short (3-4 residues). The physical effect arising from these short linkers is not known; such linkers may lead to a stiff elongated molecule or, conversely, may lead to a more compact and dynamic structure. In an effort to better understand how linkers affect obscurin flexibility, and to better understand the physical underpinnings of this flexibility, here we study the structure and dynamics of four representative sets of dual obscurin Ig domains using experimental and computational techniques. We find in all cases tested that tandem obscurin Ig domains interact at the poles of each domain and tend to stay relatively extended in solution. NMR, SAXS, and MD simulations reveal that while tandem domains are elongated, they also bend and flex significantly. By applying this behavior to a simplified model, it becomes apparent obscurin can link targets more than 200 nm away. However, as targets get further apart, obscurin begins acting as a spring and requires progressively more energy to further elongate.

At the heart of inter- and intracellular signaling: the intercalated disc.

Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.

Corrigendum: The Phosphorylation Profile of Myosin Binding Protein-C Slow is Dynamically Regulated in Slow-Twitch Muscles in Health and Disease.

This corrects the article DOI: 10.1038/srep12637.

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

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

TGF-ß1 affects cell-cell adhesion in the heart in an NCAM1-dependent mechanism.

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFß1). While TGFß1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFß1 were generated to model the observed increase in activity in the failing heart. Activation of TGFß1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFß1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFß1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFß1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFß1 activity in the human cardiac ventricle. Overall, we found that TGFß1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFß1 and reduce heart failure severity.

Novel obscurins mediate cardiomyocyte adhesion and size via the PI3K/AKT/mTOR signaling pathway.

The intercalated disc of cardiac muscle embodies a highly-ordered, multifunctional network, essential for the synchronous contraction of the heart. Over 200 known proteins localize to the intercalated disc. The challenge now lies in their characterization as it relates to the coupling of neighboring cells and whole heart function. Using molecular, biochemical and imaging techniques, we characterized for the first time two small obscurin isoforms, obscurin-40 and obscurin-80, which are enriched at distinct locations of the intercalated disc. Both proteins bind specifically and directly to select phospholipids via their pleckstrin homology (PH) domain. Overexpression of either isoform or the PH-domain in cardiomyocytes results in decreased cell adhesion and size via reduced activation of the PI3K/AKT/mTOR pathway that is intimately linked to cardiac hypertrophy. In addition, obscurin-80 and obscurin-40 are significantly reduced in acute (myocardial infarction) and chronic (pressure overload) murine cardiac-stress models underscoring their key role in maintaining cardiac homeostasis. Our novel findings implicate small obscurins in the maintenance of cardiomyocyte size and coupling, and the development of heart failure by antagonizing the PI3K/AKT/mTOR pathway.

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.

Obscure functions: the location-function relationship of obscurins.

The obscurin family of polypeptides is essential for normal striated muscle function and contributes to the pathogenesis of fatal diseases, including cardiomyopathies and cancers. The single mammalian obscurin gene, OBSCN, gives rise to giant (∼800 kDa) and smaller (∼40-500 kDa) proteins that are composed of tandem adhesion and signaling motifs. Mammalian obscurin proteins are expressed in a variety of cell types, including striated muscles, and localize to distinct subcellular compartments where they contribute to diverse cellular processes. Obscurin homologs in Caenorhabditis elegans and Drosophila possess a similar domain architecture and are also expressed in striated muscles. The long sought after question, "what does obscurin do?" is complex and cannot be addressed without taking into consideration the subcellular distribution of these proteins and local isoform concentration. Herein, we present an overview of the functions of obscurins and begin to define the intricate relationship between their subcellular distributions and functions in striated muscles.

Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles.

Myosin Binding Protein-C slow (sMyBP-C), encoded by MYBPC1, comprises a family of regulatory proteins of skeletal muscles that are phosphorylated by PKA and PKC. MYBPC1 missense mutations are linked to the development of Distal Arthrogryposis-1 (DA-1). Although structure-function details for this myopathy are evolving, function is undoubtedly driven by sequence variations and post-translational modifications in sMyBP-C. Herein, we examined the phosphorylation profile of sMyBP-C in mouse and human fast-twitch skeletal muscles. We used Flexor Digitorum Brevis (FDB) isolated from young (~2-months old) and old (~14-months old) wild type and mdx mice, and human Abductor Hallucis (AH) and gastrocnemious muscles carrying the DA-1 mutations. Our results indicate both constitutive and differential phosphorylation of sMyBP-C in aged and diseased muscles. We report a 7-35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx FDB mouse muscles, compared to young wild type tissue. Similarly, we observe a 30-70% decrease in the phosphorylation levels of all PKA and PKC phospho-sites in the DA-1 AH, but not gastrocnemius, muscle. Overall, our studies show that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age and disease, suggesting that phosphorylation plays important roles in these processes.

The Phosphorylation Profile of Myosin Binding Protein-C Slow is Dynamically Regulated in Slow-Twitch Muscles in Health and Disease.

Myosin Binding Protein-C slow (sMyBP-C) is expressed in skeletal muscles where it plays structural and regulatory roles. The functions of sMyBP-C are modulated through alternative splicing and phosphorylation. Herein, we examined the phosphorylation profile of sMyBP-C in mouse slow-twitch soleus muscle isolated from fatigued or non-fatigued young (2-4-months old) and old (~14-months old) wild type and mdx mice. Our findings are two-fold. First, we identified the phosphorylation events present in individual sMyBP-C variants at different states. Secondly, we quantified the relative abundance of each phosphorylation event, and of sMyBP-C phospho-species as a function of age and dystrophy, in the presence or absence of fatigue. Our results revealed both constitutive and differential phosphorylation of sMyBP-C. Moreover, we noted a 10-40% and a 25-35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx soleus muscles, respectively. On the contrary, we observed a 5-10% and a 20-25% increase in the phosphorylation levels of specific sites in young fatigued wild type and mdx soleus muscles, respectively. Overall, our studies showed that the phosphorylation pattern of sMyBP-C is differentially regulated following reversible (i.e. fatigue) and non-reversible (i.e. age and disease) (patho)physiological stressors.

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.

Loss of actomyosin regulation in distal arthrogryposis myopathy due to mutant myosin binding protein-C slow.

Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 µm/s at 0.1 µM, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.

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.

Myosin binding protein-C slow: a multifaceted family of proteins with a complex expression profile in fast and slow twitch skeletal muscles.

Myosin Binding Protein-C slow (sMyBP-C) comprises a complex family of proteins expressed in slow and fast type skeletal muscles. Similar to its fast and cardiac counterparts, sMyBP-C functions to modulate the formation of actomyosin cross-bridges, and to organize and stabilize sarcomeric A- and M-bands. The slow form of MyBP-C was originally classified as a single protein, however several variants encoded by the single MYBPC1 gene have been recently identified. Alternative splicing of the 5' and 3' ends of the MYBPC1 transcript has led to the differential expression of small unique segments interspersed between common domains. In addition, the NH2-terminus of sMyBP-C undergoes complex phosphorylation. Thus, alternative splicing and phosphorylation appear to regulate the functional activities of sMyBP-C. sMyBP-C proteins are not restricted to slow twitch muscles, but they are abundantly expressed in fast twitch muscles, too. Using bioinformatic tools, we herein perform a systematic comparison of the known human and mouse sMyBP-C variants. In addition, using single fiber westerns and antibodies to a common region of all known sMyBP-C variants, we present a detailed and comprehensive characterization of the expression profile of sMyBP-C proteins in the slow twitch soleus and the fast twitch flexor digitorum brevis (FDB) mouse muscles. Our studies demonstrate for the first time that distinct sMyBP-C variants are co-expressed in the same fiber, and that their expression profile differs among fibers. Given the differential expression of sMyBP-C variants in single fibers, it becomes apparent that each variant or combination thereof may play unique roles in the regulation of actomyosin cross-bridges formation and the stabilization of thick filaments.

Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.

Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca(2+) channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca(2+) were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.

Myosin binding protein-C: a regulator of actomyosin interaction in striated muscle.

Myosin-Binding protein-C (MyBP-C) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges, via direct interactions with both thick myosin and thin actin filaments. Three distinct MyBP-C isoforms have been characterized; cardiac, slow skeletal, and fast skeletal. Numerous mutations in the gene for cardiac MyBP-C (cMyBP-C) have been associated with familial hypertrophic cardiomyopathy (FHC) and have led to increased interest in the regulation and roles of the cardiac isoform. This review will summarize our current knowledge on MyBP-C and its role in modulating contractility, focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles.