Genomics-First Evaluation of Heart Disease Associated With Titin-Truncating Variants.

BACKGROUND: Truncating variants in the Titin gene (TTNtvs) are common in individuals with idiopathic dilated cardiomyopathy (DCM). However, a comprehensive genomics-first evaluation of the impact of TTNtvs in different clinical contexts, and the evaluation of modifiers such as genetic ancestry, has not been performed. METHODS: We reviewed whole exome sequence data for >71 000 individuals (61 040 from the Geisinger MyCode Community Health Initiative (2007 to present) and 10 273 from the PennMedicine BioBank (2013 to present) to identify anyone with TTNtvs. We further selected individuals with TTNtvs in exons highly expressed in the heart (proportion spliced in [PSI] >0.9). Using linked electronic health records, we evaluated associations of TTNtvs with diagnoses and quantitative echocardiographic measures, including subanalyses for individuals with and without DCM diagnoses. We also reviewed data from the Jackson Heart Study to validate specific analyses for individuals of African ancestry. RESULTS: Identified with a TTNtv in a highly expressed exon (hiPSI) were 1.2% individuals in PennMedicine BioBank and 0.6% at Geisinger. The presence of a hiPSI TTNtv was associated with increased odds of DCM in individuals of European ancestry (odds ratio [95% CI]: 18.7 [9.1-39.4] {PennMedicine BioBank} and 10.8 [7.0-16.0] {Geisinger}). hiPSI TTNtvs were not associated with DCM in individuals of African ancestry, despite a high DCM prevalence (odds ratio, 1.8 [0.2-13.7]; P=0.57). Among 244 individuals of European ancestry with DCM in PennMedicine BioBank, hiPSI TTNtv carriers had lower left ventricular ejection fraction (ß=-12%, P=3×10-7), and increased left ventricular diameter (ß=0.65 cm, P=9×10-3). In the Geisinger cohort, hiPSI TTNtv carriers without a cardiomyopathy diagnosis had more atrial fibrillation (odds ratio, 2.4 [1.6-3.6]) and heart failure (odds ratio, 3.8 [2.4-6.0]), and lower left ventricular ejection fraction (ß=-3.4%, P=1×10-7). CONCLUSIONS: Individuals of European ancestry with hiPSI TTNtv have an abnormal cardiac phenotype characterized by lower left ventricular ejection fraction, irrespective of the clinical manifestation of cardiomyopathy. Associations with arrhythmias, including atrial fibrillation, were observed even when controlling for cardiomyopathy diagnosis. In contrast, no association between hiPSI TTNtvs and DCM was discerned among individuals of African ancestry. Given these findings, clinical identification of hiPSI TTNtv carriers may alter clinical management strategies.

Tetraspanins are involved in Schwann cell-axon interaction.

Many studies have shown that tetraspanins play important role in cell-cell and cell-extracellular matrix (ECM) interactions. The repertoire and functions of tetraspanins in Schwann cells, glial cells of the peripheral nervous system have remained largely uncharacterized. This study was undertaken to identify Schwann cell tetraspanins and to elucidate their possible functions. Microarray analysis revealed the expression of numerous tetraspanins in primary culture of Schwann cells. Expression of five of them, CD9, CD63, CD81, CD82, and CD151, and of tetraspanin-associated protein EWI-2 was also confirmed by immunofluorescence. Localization of CD9, CD63, CD81, and EWI-2 was largely confined to paranodes and Schmidt-Lanterman incisures, regions of noncompact myelin. Immunoprecipitation experiments showed that these four proteins form a complex in Schwann cells. siRNA silencing of individual components of the complex did not affect Schwann cell adhesion to ECM proteins or attachment to and alignment with axons. However, suppression of both CD63 and CD81 expression together significantly inhibited extension of Schwann cell processes along axons, without affecting initial attachment of the cells to the axonal surface. Adhesion, spreading, and migration of Schwann cells on ECM proteins also were not affected by double silencing of CD63 and CD81. Suppression of CD63 and CD81 expression did not affect the ability of Schwann cells to myelinate dorsal root ganglion neurons in vitro. These findings strongly suggest that CD63 and CD81 play an important role in Schwann cell spreading along axons but seem to be dispensable for Schwann cell myelination.

Fibrillin-2 is dispensable for peripheral nerve development, myelination and regeneration.

The extracellular matrix of peripheral nerve is formed from a diverse set of macromolecules, including glycoproteins, collagens and proteoglycans. Recent studies using knockout animal models have demonstrated that individual components of the extracellular matrix play a vital role in peripheral nerve development and regeneration. In this study we identified fibrillin-1 and fibrillin-2, large modular structural glycoproteins, as components of the extracellular matrix of peripheral nerve. Previously it was found that fibrillin-2 null mice display joint contractures, suggesting a possible defect of the peripheral nervous system in these animals. Close examination of the peripheral nerves of fibrillin-2 deficient animals described here revealed some structural abnormalities in the perineurium, while general structure of the nerve and molecular composition of nerve extracellular matrix remained unchanged. We also found that in spite of the obvious motor function impairment, fibrillin-2 null mice failed to display changes of nerve conduction properties or nerve regeneration capacity. Based on the data obtained we can conclude that peripheral neuropathy should be excluded as the cause of the impairment of locomotory function and joint contractures observed in fibrillin-2 deficient animals.

Alpha7beta1 integrin is a receptor for laminin-2 on Schwann cells.

The Schwann cell basal lamina acts as an organizer of peripheral nerve tissue and influences many aspects of cell behavior during development and regeneration. A principal component of the Schwann cell basal lamina is laminin-2. This study was undertaken to identify Schwann cell receptors for laminin-2. We found that among several Schwann cell integrins that can potentially interact with laminin-2, only alpha7beta1 bound to laminin-2-Sepharose. Dystroglycan, a non-integrin Schwann cell receptor for laminin-2 identified previously, was also found to bind to laminin-2-Sepharose. Antibody to the alpha7 integrin subunit partially inhibited Schwann cell adhesion to laminin-2. Small interfering RNA-mediated suppression of either alpha7 integrin or dystroglycan expression decreased adhesion and spreading of Schwann cells on laminin-2, whereas knocking down both proteins together inhibited adhesion and spreading on laminin-2 almost completely. alpha7 integrin and dystroglycan both colocalized with laminin-2 containing basal lamina tubes in differentiating neuron-Schwann cell cocultures. The alpha7beta1 integrin also coprecipitates with focal adhesion kinase in differentiating cocultures. These findings strongly suggest that alpha7beta1 integrin is a Schwann cell receptor for laminin-2 that provides transmembrane linkage between the Schwann cell basal lamina and cytoskeleton.

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger.

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

Neuregulin isoforms in dorsal root ganglion neurons: effects of the cytoplasmic domain on localization and membrane shedding of Nrg-1 type I.

Embryonic sensory neurons express membrane-anchored growth factors that stimulate proliferation and differentiation of Schwann cells. The most important of these are members of the neuregulin-1 (Nrg-1) family that activate the erbB2/erbB3 receptor kinase on Schwann cells. Nrg-1 growth factors display a complex pattern of alternative mRNA splicing. We investigated the expression of the Nrg-1 type I in rat embryo dorsal root ganglion (DRG) neurons. Nrg-1 type I mRNA was abundantly expressed in DRG neurons; molecular cloning identified three distinct isoforms. The most prominent structural difference produced by alternative splicing was truncation of the C-terminal cytoplasmic domain. In sensory neurons and other cells, Nrg-1 type I proteins with the full-length 374-amino-acid cytoplasmic domain were expressed on the cell surface. In contrast, an isoform with a partially truncated cytoplasmic domain was retained in an intracellular compartment. Deletion studies demonstrated the presence of a cryptic intracellular retention signal that was exposed in the truncated cytoplasmic domain. Cell surface Nrg-1 type I molecules were subject to protease-dependent release of the biologically active ectodomain. As a consequence of their intracellular localization, the Nrg-1 type I isoform with a truncated cytoplasmic domain was not subject to membrane shedding. Nrg-1 type I ectodomain release was accelerated by factors present in Schwann cell-conditioned medium. In cells with active Nrg-1 type I ectodomain, shedding products corresponding to the cytoplasmic domain were not detected, because of rapid gamma-secretase- and proteasome-dependent degradation. These results demonstrate that sensory neurons express alternatively spliced neuregulin polypeptides with distinct subcellular localizations and processing.

Glypican-1 and alpha4(V) collagen are required for Schwann cell myelination.

Schwann cell myelination requires interactions with the extracellular matrix (ECM) mediated by cell surface receptors. Previously, we identified a type V collagen family member, alpha4(V) collagen, which is expressed by Schwann cells during peripheral nerve differentiation. This collagen binds with high affinity to heparan sulfate through a unique binding motif in the noncollagenous N-terminal domain (NTD). The principal alpha4(V) collagen-binding protein on the Schwann cell surface is the heparan sulfate proteoglycan glypican-1. We investigated the role of alpha4(V) collagen and glypican-1 in Schwann cell terminal differentiation in cultures of Schwann cells and dorsal root ganglion neurons. Small interfering RNA-mediated suppression of glypican-1 expression decreased binding of alpha4(V)-NTD to Schwann cells, adhesion and spreading of Schwann cells on alpha4(V)-NTD, and incorporation of alpha4(V) collagen into Schwann cell ECM. In cocultures, alpha4(V) collagen coassembles with laminin on the surface of polarized Schwann cells to form tube-like ECM structures that are sites of myelination. Suppression of glypican-1 or alpha4(V) collagen expression significantly inhibited myelination. These results demonstrate an important role for these proteins in peripheral nerve terminal differentiation.

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes.

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

Constitutive release of alpha4 type V collagen N-terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparan sulfate proteoglycans.

During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain alpha4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The alpha4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the alpha4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, alpha4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the alpha4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released alpha4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major alpha4(V)-NTD-binding proteins are glypican-1 and perlecan. alpha4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains.

Specific assembly pathway of sarcoglycans is dependent on beta- and delta-sarcoglycan.

Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt sarcoglycan-complex formation. Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.

Matrix metalloproteinase-dependent shedding of syndecan-3, a transmembrane heparan sulfate proteoglycan, in Schwann cells.

Schwann cells transiently express the transmembrane heparan sulfate proteoglycan syndecan-3 during the late embryonic and early postnatal periods of peripheral nerve development. Neonatal rat Schwann cells released soluble syndecan-3 into the culture medium by a process that was blocked by inhibition of endogenous matrix metalloproteinase activity. When Schwann cells were plated on a substratum that binds syndecan-3, the released proteoglycan bound to the substratum adjacent to the cell border. Membrane-anchored syndecan-3 was concentrated in actin-containing filopodia that projected from the lateral edges of the Schwann cell membrane. Membrane shedding was specific for syndecan-3 and was not observed for the related proteoglycan syndecan-1. Analysis of Schwann cells transfected with wild-type and chimeric syndecan-1 and syndecan-3 cDNAs revealed that membrane shedding was a property of the syndecan-3 ectodomain. Inhibition of syndecan-3 release significantly enhanced Schwann cell adhesion and process extension on dishes coated with the non-collagenous N-terminal domain of alpha4(V) collagen, which binds syndecan-3 and mediates heparan sulfate-dependent Schwann cell adhesion. Matrix metalloproteinase-dependent syndecan-3 shedding was also observed in newborn rat peripheral nerve tissue. Syndecan-3 shedding in peripheral nerve tissue was age specific, and was not observed during later stages of postnatal nerve development. These results demonstrate that Schwann cell syndecan-3 is subject to matrix metalloproteinase-dependent membrane processing, which modulates the biological function of this proteoglycan.

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.

Syndecan-3 and perlecan are differentially expressed by progenitors and mature oligodendrocytes and accumulate in the extracellular matrix.

Oligodendrocyte progenitors originate in the subventricular zone, proliferate, migrate to their final destinations, differentiate, and interact with axons to produce multilamellar myelin sheaths. These processes are regulated by a variety of environmental signals, including growth factors, the extracellular matrix, and adhesion molecules. Heparan sulfate proteoglycans are premier candidates as participants in this regulation by virtue of their structural diversity and their capacity to function as coreceptors for both growth factors and extracellular matrix molecules. Consistently with this, we have previously shown that oligodendrocyte progenitors are unable to proliferate in response to fibroblast growth factor-2 (FGF-2) in the absence of sulfated heparan sulfate proteoglycan. Here we show that members of three families of heparan sulfate proteoglycans, syndecan, perlecan, and glypican, are developmentally and posttranscriptionally regulated during oligodendrocyte-lineage progression: Syndecan-3 is synthesized by oligodendrocyte progenitors (but not terminally differentiated oligodendrocytes) and is up-regulated by FGF-2; perlecan synthesis increases as oligodendrocytes undergo terminal differentiation; glypican-1 is expressed by both progenitors and differentiated oligodendrocytes. Astrocytes express glypican-1 and perlecan but not syndecan-3. All three of these heparan sulfate proteoglycans are shed from the cell surface and bind to specific substrates. The developmentally regulated expression of these heparan sulfate proteoglycans is indicative of their participation in events involving growth factor receptors and the extracellular matrix that may regulate oligodendrocyte progenitor proliferation, migration, and adhesion phenomena.

Schwann cell adhesion to a novel heparan sulfate binding site in the N-terminal domain of alpha 4 type V collagen is mediated by syndecan-3.

Previously we reported that type V collagen synthesized by Schwann cells inhibits the outgrowth of axons from rat embryo dorsal root ganglion neurons but promotes Schwann cell migration (Chernousov, M. A., Stahl, R. C., and Carey, D. J. (2001) J. Neurosci. 21, 6125-6135). Analysis of Schwann cell adhesion and spreading on dishes coated with various type V collagen domains revealed that Schwann cells adhered effectively only to the non-collagenous N-terminal domain (NTD) of the alpha4(V) collagen chain. Schwann cell adhesion to alpha4(V)-NTD induced actin cytoskeleton assembly, tyrosine phosphorylation, and activation of the Erk1/Erk2 protein kinases. Adhesion to alpha4(V)-NTD is cell type-specific because rat fibroblasts failed to adhere to dishes coated with this polypeptide. Schwann cell adhesion and spreading on alpha4(V)-NTD was strongly inhibited by soluble heparin (IC(50) approximately 30 ng/ml) but not by chondroitin sulfate. Analysis of the heparin binding activities of a series of recombinant alpha4(V)-NTD fragments and deletion mutants identified a highly basic region (not present in other type V collagen NTD) as the site responsible for high affinity heparin binding. Schwann cells adhered poorly to dishes coated with alpha4(V)-NTD that lacked the heparin binding site and failed to spread or assemble organized actin-cytoskeletal structures. Soluble alpha4(V)-NTD polypeptide that contained the heparin binding site inhibited spreading of Schwann cells on dishes coated with alpha4(V)-NTD. Affinity chromatography of Schwann cell detergent extracts on a column of immobilized alpha4(V)-NTD resulted in the isolation of syndecan-3, a transmembrane heparan sulfate proteoglycan. Together, these results suggest that Schwann cells bind to collagen type V via syndecan-3-dependent binding to a novel high affinity heparin binding site in the alpha4(V)-NTD.