Alpha 1-syntrophin Research Articles

Abstract 315: Time-resolved Expression of Membrane-targeted Proteins During Ipsc-cardiomyocyte Differentiation

Background: The ability to create human cardiomyocytes from induced pluripotent stem cells (iPSCs) has spurred advances in disease modeling and drug testing, but there remains an ongoing need to improve the purity and functional maturity of derived cardiomyocytes. Membrane proteins are an integral component of cell signaling and specification. To better understand iPSC-cardiomyocyte differentiation and identify potential surface markers of cell maturity, we set out to examine how membrane-targeted proteins gradually remodel in differentiating cardiomyocytes. Methods: Human iPSCs from three donors were differentiated into cardiomyocytes in 40 independent differentiation plates. We sampled protein expression at independent time points at up to daily intervals during day 0 to day 14 post-differentiation, then sparsely up to 100 days. The extracted proteins were analyzed using a Q-Exactive HF Orbitrap high-resolution mass spectrometer to quantify how the relative quantity of membrane-targeted proteins changes over time in differentiating cells. Results: We identified 1095 annotated cell membrane proteins and quantified their temporal changes in abundance across 40 samples. Protein expression in the first two principal components recapitulated gradual differentiation of iPSCs into mesoderm, cardiac progenitors, and more mature cardiomyocytes. Membrane protein expression partitioned the samples into three discernible clusters, representing early (day 0-5), mid (day 6-10), and late (day 11+) differentiation time points. In total, 115 cell membrane proteins showed differential expression in late vs. early differentiation, including two popeye-domain-containing proteins (POPDC2 and BVES) known to be involved in cardiac development. In particular, we identified two proteins, dysferlin (DYSF) and alpha-1 syntrophin (SNTA1), which continued to show strong upward changes in late differentiation time points, nominating these proteins for investigation as markers for mature cells. Conclusion: We report the time-resolved expression profiles of over 1000 membrane-targeted proteins during iPSC-cardiomyocyte differentiation. Future work into the membrane proteome may lead to maturity markers to guide maturation efforts.

Channelopathies and drug discovery in the postgenomic era

Ion channels are a diverse group of pore-forming proteins that provide selective pathways for the movement of ions (Na+, K+, Ca2+, Cl−, etc) across the lipid membrane barrier. Aquaporins facilitate water movement across cell membranes in response to osmotic gradients. There is an increasing body of information on the molecular structure and functional roles of ion and water channels in health and disease, linking channel function at the molecular level to organ physiology. Because ion channels play essential roles in all cells, defects in ion channels are associated with a wide variety of pathophysiological conditions and human diseases. Diseases caused by disturbed function of ion channel subunits or regulatory proteins have been defined as “channelopathies”1, 2. In the postgenomic era the rapid progress in the molecular identification of genes for new ion channels and elucidation of their transport properties, physiological functions, and disease relevant mutations has significantly advanced the knowledge of channelopathies and potential new therapies3, 4, 5, 6, 7, 8, 9. For examples, the discovery of aquaporin water channels, a family of integral membrane proteins that selectively transport water, has led to the identification of water channelopathies including autosomal dominant and recessive forms of hereditary nepherogenic diabetes insipidus caused by aquaporin-2 mutations10, congenital cataracts incurred by aquaporin-0 mutations11 and aquired neuronal inflammatory disease neuromyelitis optica in which pathogenic autoantibodies target aquaporin-412. Impaired Cl− transport caused by mutations in genes belonging to distinct Cl− channel families has been found to cause diverse diseases such as cystic fibrosis, myotonia, epilepsy, hyperekplexia, lysosomal storage disease, deafness, renal salt loss, kidney stones, osteopetrosis, and cardiovascular diseases7, 13, 14. In addition, the studies on the role of ion channel regulatory proteins, including the sub-membrane adapter ankyrins and alpha-1 syntrophin, membrane coat protein caveolin-3, signaling platform yotiao, and lamins, have also provided novel insights into understanding of human diseases15.

Beyond membrane channelopathies: alternative mechanisms underlying complex human disease

Over the past fifteen years, our understanding of the molecular mechanisms underlying human disease has flourished in large part due to the discovery of gene mutations linked with membrane ion channels and transporters. In fact, ion channel defects ("channelopathies" - the focus of this review series) have been associated with a spectrum of serious human disease phenotypes including cystic fibrosis, cardiac arrhythmia, diabetes, skeletal muscle defects, and neurological disorders. However, we now know that human disease, particularly excitable cell disease, may be caused by defects in non-ion channel polypeptides including in cellular components residing well beneath the plasma membrane. For example, over the past few years, a new class of potentially fatal cardiac arrhythmias has been linked with cytoplasmic proteins that include sub-membrane adapters such as ankyrin-B (ANK2), ankyrin-G (ANK3), and alpha-1 syntrophin, membrane coat proteins including caveolin-3 (CAV3), signaling platforms including yotiao (AKAP9), and cardiac enzymes (GPD1L). The focus of this review is to detail the exciting role of lamins, yet another class of gene products that have provided elegant new insight into human disease.

The Sarcolemmal Calcium Pump, α-1 Syntrophin, and Neuronal Nitric-oxide Synthase Are Parts of a Macromolecular Protein Complex

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and alpha-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, alpha-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399-447 of alpha-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of alpha-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels of alpha-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4-/- mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin.dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality.

The sarcolemmal calcium pump, alpha-1 syntrophin and neuronal nitric oxide synthase are part of a macromolecular protein complex

The sarcolemmal calcium pump, alpha-1 syntrophin and neuronal nitric oxide synthase are part of a macromolecular protein complex

Nitric oxide synthase in skeletal muscle fibers: a signaling component of the dystrophin-glycoprotein complex.

The present review deals with the anatomical distribution, physiological importance, and pathological implications of the neuronal-type nitric oxide synthase (nNOS) in skeletal muscle. Throughout the body, nNOS is located beneath the sarcolemma of skeletal muscle fibers. In rodents, nNOS is enriched in type IIb muscle fibers, but is more homogenously distributed among type II and type I fibers in humans and subhuman primates. It is accumulated on the postsynaptic membrane at the neuromuscular junction. An increased concentration of nNOS is noted at the sarcolemma of muscle spindle fibers, in particular nuclear bag fibers, which belong to type I fibers. The association of nNOS with the sarcolemma is mediated by the dystrophin-glycoprotein complex. Specifically, nNOS is linked to alpha 1-syntrophin through PDZ domain interactions. Possibly, it also directly binds to dystrophin. The activity and expression of nNOS are regulated by both myogenic and neurogenic factors. Besides acetylcholine, glutamate has also been shown to stimulate nNOS, probably acting through N-methyl-D-aspartate receptors, which are colocalized with nNOS at the junctional sarcolemma. Functional studies have implicated nitric oxide as a modulator of skeletal muscle contractility, mitochondrial respiration, carbohydrate metabolism, and neuromuscular transmission. A clinically relevant aspect of nNOS is its absence from the skeletal muscle sarcolemma of patients with Duchenne muscular dystrophy (DMD). A concept is presented which suggests that, as a consequence of the disruption of the dystrophin-glyoprotein complex in DMD, nNOS fails to become attached to the sarcolemma and is subject to downregulation in the cytosol.

Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions.

Ultrastructural localization of alpha 1-syntrophin and neuronal nitric oxide synthase in normal skeletal myofiber, and their relation to each other and to dystrophin.

We investigated the ultrastructural localization of alpha 1-syntrophin and neuronal nitric oxide synthase (nNOS) in normal human skeletal myofibers and analyzed their relation to each other and to dystrophin using single and double immunogold-labeling electron microscopy. Single immunolabeling showed antibodies to alpha 1-syntrophin and nNOS on the inner surface of the muscle plasma membrane, the sarcoplasmic side of plasma membrane invaginations, and the sarcoplasm near mitochondria of subsarcolemmal areas. The epitopes of alpha 1-syntrophin and nNOS tended to be present in clusters. Double immunolabeling revealed that epitope combinations of alpha 1-syntrophin-dystrophin, alpha 1-syntrophin-nNOS, and nNOS-dystrophin occurred more frequently in doublet form than did other epitope combinations, such as alpha 1-syntrophin-beta- spectrin and nNOS-beta-spectrin. These increased frequencies were noted both at the muscle plasma membrane undercoat and near mitochondria of subsarcolemmal areas. A significantly higher percentage of doublets comprised antibodies against alpha 1-syntrophin and dystrophin (28.5 +/- 1.5%, group mean +/- SE) than those against alpha 1-syntrophin and beta-spectrin (9.2 +/- 0.8%, P < 0.01). Furthermore, nNOS formed doublets significantly more frequently with dystrophin (25.2 +/- 3.3%) and alpha 1-syntrophin (26.0 +/- 4.1%) than with beta-spectrin (13.9 +/- 2.3%; P < 0.05). These data support the association of dystrophin, alpha 1-syntrophin, and nNOS at the inner surface of the muscle plasma membrane and near mitochondria of subsarcolemmal areas of normal human skeletal myofibers.

Ca2+-calmodulin binding to mouse alpha1 syntrophin: syntrophin is also a Ca2+-binding protein.

Syntrophins are peripheral membrane proteins which have been found associated with dystrophin, the protein product of the Duchenne muscular dystrophy gene locus. Mouse alpha1 syntrophin binds the COOH-terminal domain of dystrophin, and calmodulin inhibits this interaction in a Ca2+-dependent fashion. Where calmodulin binds to syntrophin was investigated by constructing fusion proteins containing different regions of syntrophin's sequence. Syntrophin contains at least two regions which bind calmodulin in different ways. The COOH-terminal 24 residues contain a Ca2+-calmodulin binding site, named CBS-C, which binds calmodulin with an apparent affinity of 18 nM and which is highly conserved in all syntrophins. The amino-terminal 174 residue section of syntrophin contains other calmodulin binding, and binding occurs in either the presence or absence of Ca2+ with an apparent affinity of 100 nM. Syntrophin was shown to bind Ca2+ at two or more sites residing in the amino-terminal 274 residues, and Ca2+ binding to syntrophin affects calmodulin binding at high concentrations of syntrophin. Syntrophin A (residues 4-274) is predominantly a dimer in EGTA. A model of syntrophin's complex interactions with itself (i.e., oligomerization), calmodulin, and Ca2+ is presented.

Absence of nitric oxide synthase I despite the presence of the dystrophin complex in human striated muscle.

Recently, it has been shown that in human striated muscle the signalling enzyme, brain-type nitric oxide synthase I (NOS I), is associated with the sarcolemma and complexes with dystrophin and/or members of the dystrophin complex. In order to find out whether there exists a regular association between NOS I and the complex, muscle biopsies from patients with various muscle disorders were analysed by enzyme histochemistry and immunohistochemistry. In patients suffering from Duchenne muscular dystrophy, and to a lesser extent in those with Becker-type dystrophy, NOS I and dystrophin complex components were absent or drastically reduced in the sarcolemma region. In other dystrophies, as well as in metabolic and inflammatory myopathies, NOS I and dystrophin complex constituents were expressed normally, while in the case of neurogenic diseases leading to denervation atrophy and especially congenital idiopathic clubfoot, the immunohistochemical patterns of the distribution of the dystrophin complex constituents were normal, but NOS I activity and protein were deficient or dramatically diminished. The results can be interpreted as indicating that, in general, NOS I targeting to the sarcolemma is dependent on particular members of the dystrophin complex, such as alpha-1 syntrophin, yet the expression and/or positioning of NOS I may be under the control of further factors, probably of neurogenic origin. NOS I-associated diaphorase may thus be a useful complementary tool in the diagnosis of muscle disorders.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

The Three Human Syntrophin Genes Are Expressed in Diverse Tissues, Have Distinct Chromosomal Locations, and Each Bind to Dystrophin and Its Relatives

The syntrophins are a biochemically heterogeneous group of 58-kDa intracellular membrane-associated dystrophin-binding proteins. We have cloned and characterized human acidic (alpha 1-) syntrophin and a second isoform of human basic (beta 2-) syntrophin. Comparison of the deduced amino acid structure of the three human isoforms of syntrophin (together with the previously reported human beta 1-syntrophin) demonstrates their overall similarity. The deduced amino acid sequences of human alpha 1- and beta 2-syntrophin are nearly identical to their homologues in mouse, suggesting a strong functional conservation among the individual isoforms, Much like beta 1-syntrophin, human beta 2-syntrophin has multiple transcript classes and is expressed widely, although in a distinct pattern of relative abundance. In contrast, human alpha 1-syntrophin is most abundant in heart and skeletal muscle, and less so in other tissues. Somatic cell hybrids and fluorescent in situ hybridization were both used to determine their chromosomal locations: beta 2-syntrophin to chromosome 16q22-23 and alpha 1-syntrophin to chromosome 20q11.2. Finally, we used in vitro translated proteins in an immunoprecipitation assay to show that, like beta 1-syntrophin, both beta 2- and alpha 1-syntrophin interact with peptides encoding the syntrophin-binding region of dystrophin, utrophin/dystrophin related protein, and the Torpedo 87K protein.

Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice-prone region of the dystrophin COOH terminus.

The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283-292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1-syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin-associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.